19.1.6: Arabidopsis Thaliana - A Model Organism - Biology

19.1.6: Arabidopsis Thaliana - A Model Organism - Biology

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Arabidopsis Thaliana has become to plant biology what Drosophila melanogaster and Caenorhabditis elegans are to animal biology. Arabidopsis is an angiosperm, a dicot from the mustard family (Brassicaceae). It is popularly known as thale cress or mouse-ear cress. While it has no commercial value - in fact is considered a weed - it has proved to be an ideal organism for studying plant development.

Some of its advantages as a model organism:

  • It has one of the smallest genomes in the plant kingdom: 135 x 106 base pairs of DNA distributed in 5 chromosomes (2n = 10) and almost all of which encodes its 27,407 genes.
  • Transgenic plants can be made easily using Agrobacterium tumefaciens as the vector to introduce foreign genes.
  • The plant is small - a flat rosette of leaves from which grows a flower stalk 6–12 inches high.
  • It can be easily grown in the lab in a relatively small space.
  • Development is rapid. It only takes 5– 6 weeks from seed germination to the production of a new crop of seeds.
  • It is a prolific producer of seeds (up to 10,000 per plant) making genetics studies easier.
  • Mutations can be easily generated (e.g., by irradiating the seeds or treating them with mutagenic chemicals).
  • It is normally self-pollinated so recessive mutations quickly become homozygous and thus expressed.

Other members of its family cannot self-pollinate. They have an active system of self-incompatibility. Arabidopsis, however, has inactivating mutations in the genes — SRK and SCR - that prevent self-pollination in other members of the family.

  • However, Arabidopsis can easily be cross-pollinated to do genetic mapping and produce strains with multiple mutations.

Many of the findings about how plants work - described throughout these pages - were learned from studies with Arabidopsis.

Basic leucine zipper 19

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Materials and Methods

Plant Material and Genotyping

The A. thaliana (L.) Heynh. SALK and SAIL T-DNA insertion lines in ecotype Columbia (Col-0) were obtained from the Salk Institute, Genomic Analysis Laboratory 1 (Alonso et al., 2003) and from the Syngenta collection of T-DNA insertion mutants (Sessions et al., 2002), respectively. The GABI T-DNA mutants (GK in Col-0) were generated in the context of the GABI-Kat program (MPI for Plant Breeding Research, Cologne, Germany 2 Rosso et al., 2003). All lines were provided by the Nottingham Arabidopsis Stock Centre 3 .

Seeds were germinated in soil followed by cultivation under short day conditions (8 h light/16 h dark) at 18ଌ. After 1 month the plants were transferred to long day conditions (16 h light, 22ଌ/8 h dark, 21ଌ). Genomic DNA was isolated from rosette leaves and used for PCR-based genotyping to identify heterozygous and homozygous T-DNA insertion mutants. The PCR primers used for genotyping are listed in Supplementary Table S1, and their positions are shown in Figure 1B . The following PCR program was used: initial denaturation for 5 min at 95ଌ, then 40 cycles with 15 s denaturation at 95ଌ, 30 s annealing at 55ଌ, and 60 s final elongation at 72ଌ.

Polymerase chain reaction using the gene-specific primer sets yielded DNA fragments of 𢏁 kb representing the wild-type alleles. The PCR fragments specific for the disrupted allele yielded PCR products of 𢏀.5 kb. The positions of T-DNA insertion were confirmed by sequencing the PCR-amplified T-DNA junction fragments (Supplementary Table S2).

To obtain double T-DNA insertion mutants cross-fertilization was performed.

Brassica rapa L. plants were grown under long day conditions (16 h light, 22ଌ/8 h dark, 18ଌ) to obtain meiocytes for immunolocalization of NSE4A via specific antibodies.

In silico Analysis of Gene and Protein Structures and the Phylogenetic Tree Construction

Gene structures of NSE4A and NSE4B were predicted at (Version 10 4 , 5 ). The conserved functional domains of known putative NSE4 orthologs of higher plants (full-length sequences are available at were identified using the Conserved Domain Database 6 . The same sequences were used to generate a phylogenetic tree by Bayesian phylogenetic inference in MrBayes 3.2.6 7 . All alignments were performed by the Clustal Omega 2.1 software 8 .

Gene Expression Analysis

Total RNA was isolated from seedlings, three and 6 weeks old leaves, flower buds, and root tissues using the Trizol (Thermo Fisher Scientific) method according to manufacturer’s instructions. Then, the samples were DNase-treated applying the TURBO DNA-free TM Kit (Thermo Fisher Scientific). Reverse transcription (RT) was performed using the random hexamer RevertAid Reverse Transcriptase Kit (Thermo Fisher Scientific). After 5 min initial denaturation at 95ଌ, followed by 60 min cDNA synthesis at 42ଌ, the reaction was terminated at 70ଌ for 5 min.

Quantitative real-time PCR with SYBR Green was performed using a QuantStudio 5 flex machine and the QuantStudio TM Real-Time PCR Software (v1.1). One microliter of cDNA was applied for each reaction with three replicates and three independent biological repetitions for each tissue or developmental stage. The following PCR program was used: initial denaturation for 5 min at 95ଌ, then 40 cycles with 15 s denaturation at 95ଌ, 30 s annealing at 60ଌ, and 20 s final elongation at 72ଌ. PP2A (AT1G13320) and RHIP1 (AT4G26410) served as standards (Czechowski et al., 2005). Calculations were based on the delta CT values of the reference genes (Livak and Schmittgen, 2001). The quantitative real-time RT-PCR primers used to amplify transcripts are shown in Figure 1B and Supplementary Table S3.

Cloning and Transformation

PCR-based amplification of cDNA (for 35S::Nse4A::EYFP) and genomic DNA (for promoterNse4A::gNse4A::GFP) as templates were performed using the KOD Xtreme TM Hot Start DNA Polymerase (Merck). The PCR products were cloned into the pJET 1.2 vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific). Sequence-confirmed inserts were cloned into the Gateway ® pENTR TM 1A Dual Selection Vector (Thermo Fisher Scientific). Next, the inserts were re-cloned into the pGWG (complementation vector without promoter and tag), pGWB642 (35S promoter with EYFP tag on N-term) and pGWB604 (no promoter, GFP-tag on C-terminus) vectors (Neyagawa vectors, Nakamura et al., 2010) using the BP Clonase II kit (Gateway ® Technology, Thermo Fisher Scientific). The binary vectors were transferred into Agrobacterium tumefaciens, and then used to transform A. thaliana Col-0 wild-type plants via the floral dip method (Clough and Bent, 1998). Seeds from these plants were propagated on PPT medium (16 μg/ml). Positively selected seedlings were transferred into soil and genotyped for the presence of the construct. Homozygous F2 plants were used in further studies. Primers used for the cloning are listed in Supplementary Table S4.

Recombinant Protein and Antibody Production

For antibody production the partial NSE4A peptide (from 49 to 289 aa) (Supplementary Figure S1) was expressed in the E. coli BL21 pLysS strain using the pET23a (Novagen) vector. Primers used for the recombinant protein production are listed in Supplementary Table S4. The recombinant proteins containing 6xHis-tags were purified using Dynabeads His-Tag (Thermo Fisher Scientific) according to manufacturer’s instructions. Five hundred microliters cleared extract was mixed with 500 μl binding buffer (50 mM NaP, pH 8.0, 300 mM NaCl, 0.01% Tween-20), and 50 μl washed Dynabeads were added. After 10 min incubation on a roller, the beads were washed 7 × with binding buffer, and 7 × with binding buffer, 5 mM imidazole. The elution was done with binding buffer, 150 mM imidazole, and the protein concentration (90 ng/μl) was determined using a Bradford kit (Bio-Rad Laboratories GmbH, Munich) (Bradford, 1976).

The separation on SDS gels and the protein size determination by Western analysis was done as described (Conrad et al., 1997 Supplementary Figure S2A).

Two rabbits were immunized with 1 mg NSE4A protein and complete Freund’s adjuvants. Four and five weeks later, booster immunizations were performed with 0.5 mg NSE4A protein and incomplete Freund’s adjuvants, respectively. Ten days later blood was taken, serum isolated, precipitated in 40% saturated ammonium sulfate, dialysed against 1 × PBS and affinity purified.

The specific binding behavior of the rabbit anti-NSE4 antibodies was investigated by competitive ELISA according to Conrad et al. (2011). The wells were coated with 46 ng/100 μl recombinant affinity-purified NSE4A in 1 × PBS and incubated overnight at room temperature. After blocking with 3% w/v BSA in 1 × PBS-0.05% w/v Tween 20 (1 × PBS-T) for 2 h, the known amounts of affinity-purified anti-NSE4A antibodies were mixed with various concentrations of NSE4A in 1% w/v BSA in 1 × PBS-T, incubated for 30 min in a master plate, added to the antigen-coated wells and incubated for 1 h at 25ଌ. Antibodies bound to the plate were visualized with anti-rabbit-IgG alkaline phosphatase diluted in 1 × PBS-T/1% BSA. The enzymatic substrate was pNP phosphate, and the absorbance (405 nm) was measured after 30 min incubation at 37ଌ (Supplementary Figure S2B).

To further prove the NSE4A antibody specificity in immuno-histological experiments antigen competition experiments were performed. NSE4A was added to the antibodies at a concentration of 800 nM, and applied to flow-sorted 8C A. thaliana interphase nuclei. The signal reduction compared to the control nuclei without addition of antigen clearly confirmed the specificity (Supplementary Figure S2C).

Complementation Assay

To confirm that the phenotypes of the of Nse4A mutant GK-768H08 are indeed caused by this mutation we complemented the mutant by the genomic wild-type Nse4A gene. The genomic intron-exon containing Nse4A gene with a 1.7 kbp-long upstream promoter region was amplified by PCR using the KOD Xtreme TM Hot Start DNA Polymerase (Merck), and then sequenced. Next, it was cloned into the pBWG vector (Nakamura et al., 2010), and transformed into A. tumefaciens. Plant transformation was performed by the bacteria-mediated vector transfer via the floral dipping method (Clough and Bent, 1998), and afterward propagated under long-day conditions. The harvested seeds were grown on selective PPT medium (16 μg/ml), and positively selected seedlings were transferred to soil and genotyped for the presence of the construct. Homozygous F2 plants were used in further studies.

Fertility Evaluation and Alexander Staining

Mature dry siliques were collected to evaluate silique length and seed setting. The seeds were classified into normal and shriveled ( Figure 2 ). For clearing, fully developed green siliques were treated in an ethanol:acetic acid (9:1) solution overnight at room temperature, then washed in 70 and 90% ethanol for 5 min each, followed by storage in a chloral hydrate:glycerol:water (8:1:3) solution at 4ଌ.

Impaired growth and fertility of nse4 mutants compared to wild-type (wt). (A) Reduced plant size of the mutants GK-768H08 and the double mutant GK-768H08/SAIL_296_F02. Mutant SALK_057130 and the complemented GK-768H08 mutant show a wild-type habit. (B) Reduced seed set per silique in the nse4A and nse4B mutants. (C) Shriveled seeds (arrows) of the GK-768H08 mutant. (D) Reduced pollen grain number and aborted pollen grains in an anther of the double mutant GK-768H08/SAIL_296_F02.

To evaluate anther shape and pollen viability, Alexander staining (Alexander, 1969) was performed. Undamaged anthers were used for total pollen (per anther) counting. Afterward, the anthers were squashed and the released pollen grains were evaluated into two classes: normal (viable, pink round grains), and aborted (gray/green abnormal shape).

Images from siliques, seeds and anthers were acquired using a Nikon SMZ1500 binocular and the NIS-Elements AR 3.0 software.

Bleomycin Treatment

To induce DNA DSBs via bleomycin application A. thaliana wild-type and NSE4A mutant seeds were sterilized 10 min in 70% ethanol, then 15 min in 4% Na-hypochlorite + 1 drop Tween-20, followed by washing 3 × 5 min in sterile water. The seeds were germinated on wet filter paper for 5 days, and then placed in liquid germination medium (Murashige and Skoog, Duchefa, prod. no. M0231.0025 10 g/l sucrose, 500 mg/l MES, pH 5.7) without and with bleomycin (bleomycin sulfate from Streptomyces verticillus, Sigma, cat. no. 15361) of increasing concentration. Accordingly, in a second experiment the sterilized seeds were grown on agar plates (germination medium + 2% agar-agar Roth, cat. no. 2266.2) without and with bleomycin. Both experiments were repeated twice and contained two repetitions.

Immunostaining and FISH

Flower bud fixation, chromosome slide preparation, and FISH followed by chiasma counting were performed according to Sánchez-Morán et al. (2001). To identify individual chromosomes, 5S and 45S rDNA FISH was performed.

Fluorescence in situ hybridization with telomere- and centromere-specific probes was applied to identify chromosomes at metaphase I. The 180-bp centromeric repeat probe (pAL) (Martinez-Zapater et al., 1986) was generated by PCR as previously described (Kawabe and Nasuda, 2005). The telomere-specific probe was generated by PCR in the absence of template DNA using the primers (TAAACCC)7 and (GGGTTTA)7 (Ijdo et al., 1991).

Immunostaining of A. thaliana and B. rapa PMCs followed the protocol of Armstrong and Osman (2013). The following primary antibodies were applied: rabbit anti-NSE4A (1:250) and rat anti-ZYP1 (1:1000 kindly provided by Chris Franklin). ZYP1 is the A. thaliana transverse filament protein of the synaptonemal complex (Higgins et al., 2005). The primary antibodies were detected by donkey anti-rabbit-Alexa488 (Dianova, no. 711545152) and goat anti-rat-DyLight594 (Abcam, no. ab98383), respectively, as secondary antibodies.

8C leaf interphase nuclei were flow sorted according to Weisshart et al. (2016), and also immuno-labeled against NSE4A as described above.


To image fixed and live cell preparations an Olympus BX61 microscope (Olympus) and a confocal laser scanning microscope LSM 780 (Carl Zeiss GmbH), respectively, were used.

To analyze the ultrastructure of immunosignals and chromatin beyond the classical Abbe/Raleigh limit at a lateral resolution of � nm (super-resolution, achieved with a 488 nm laser) spatial structured illumination microscopy (3D-SIM) was applied using a 63 × 1.4NA Oil Plan-Apochromat objective of an Elyra PS.1 microscope system and the software ZEN (Carl Zeiss GmbH). Images were captured separately for each fluorochrome using the 561, 488, and 405 nm laser lines for excitation and appropriate emission filters (Weisshart et al., 2016).


Two Conserved Nse4 Genes Are Present and Expressed in A. thaliana

According to previous SMC5/6 subunit prediction studies (Schubert, 2009) A. thaliana encodes two Nse4 homologs: Nse4A (AT1G51130) and Nse4B (AT3G20760) ( Figure 1A,B ). Both NSE4 proteins show similar lengths (NSE4A: 403 aa NSE4B: 383 aa), and a high amino acid sequence identity (67.7%) (Supplementary Figure S1). Both A. thaliana NSE4 proteins show similar lengths as those of budding yeast (402 aa), mouse (381 aa for NSE4A 375 aa for NSE4B), and human NSE4A (385 aa), but are longer than the fission yeast NSE4 (300 aa) and the human NSE4B (333 aa) proteins (NSE4A 9 NSE4B 10 ).

NSE4A shows a relatively high amino acid similarity compared to both B. rapa putative NSE4 proteins (Supplementary Figure S3), and other plant species (Supplementary Figure S4A). Non-plant organisms such as fission yeast, Entamoeba, Dictyostelium, mouse and human display a lower similarity (Supplementary Table S5).

The phylogenetic analysis of the full-length protein sequences of eudicot and monocot species suggests also a relatively high conservation of both A. thaliana Nse4 genes (Supplementary Figure S4B).

According to Uniprot databases 11 , both A. thaliana NSE4 proteins possess conserved C-terminal domains typical for other plant NSE4 proteins ( Figure 1C and Supplementary Figures S1, S3). The C-terminal domain binds to SMC5 in the similar way as the other kleisin molecules interact with their kappa-SMC partners (Palecek et al., 2006 Hassler et al., 2018). This interaction is crucial for the function of SMC5/6. The NSE4 N-terminal domain is also conserved and binds to SMC6 (Palecek et al., 2006). In NSE4 of fungi and vertebrates, a NSE3/MAGE binding domain was identified next to the N-terminal kleisin motif (Guerineau et al., 2012). Based on the Motif Scan analysis 12 the SMC6-binding domain can also be predicted in the NSE4 proteins of A. thaliana (Supplementary Figure S1). However, to define this identified region as the SMC6-binding motif clearly, protein–protein interaction, domain dissection and mutagenesis experiments have to be performed. Additionally, putative degradation regions and SUMOlisation sites were identified using Eukaryotic Linear Motif 13 resources (Supplementary Figure S1), suggesting that the cellular amount of NSE4 proteins during the cell cycle might be regulated via their proteolytic degradation.

In silico analysis shows a similar expression behavior (with peaks at the young rosette and flowering stages) during plant development of the Nse4A gene and other SMC5/6 subunit candidate genes, supporting a synchronized activity (Supplementary Figure S5). However, it is not clear whether they act separately or as multi-subunit complexes in various subunit combinations. In silico analysis indicated also a high co-expression of Nse4A, among others, with meiosis- and chromatin-related genes (Supplementary Table S6).

The in silico analysis of the relative expression level of Nse4A and Nse4B in ten anatomical parts of A. thaliana seedlings displayed that the expression of Nse4B is limited to generative tissues and seeds. A relatively high expression is evident only in seeds (embryo and especially endosperm) (Supplementary Figure S6).

By quantitative real-time PCR we found that Nse4A is highly expressed in flower buds and roots, but transcripts are also present in seedlings, young and old leaves (Supplementary Figure S7). In agreement with previous studies (Watanabe et al., 2009), the expression of Nse4B in these tissues is not detectable. Obviously, most Nse4B transcripts are present in already well developed seeds, as also indicated by in silico analysis (Supplementary Figure S6).

To figure out whether the NSE4 proteins interact with the other components of the SMC5/6 complex ( Figure 1 ) a protein-protein interactions analysis was performed in silico using the STRING program 14 . Interestingly, all SMC5/6 subunits accessible via the STRING program were identified as interacting partners of the NSE proteins at a very high score Ϡ.95, suggesting that both NSE4A and NSE4B act also within the SMC5/6 complex. In addition, cohesin and condensin subunits were detected as parts of the same protein-protein interaction network at the high score of Ϡ.70 (Supplementary Figure S8). An interaction with cell cycle factors could not be identified at a medium score Ϡ.5.

The results indicate that both A. thaliana Nse4 genes are highly conserved, and that the corresponding proteins may act in combination with other SMC5/6 complex components, as well as cohesin and condensin. Based on the level of expression, Nse4A seems to be the more essential gene, although Nse4B appears to be specialized to act during seed development.

Selection and Molecular Characterization of A. thaliana nse4 Mutations and Their Effect on Plant Viability, Fertility, and DNA Damage Repair

From the A. thaliana SALK, Syngenta SAIL and GABI-Kat collections, homo- and heterozygous T-DNA insertion mutants were selected for both genes ( Figure 1B and Table 1 ). The presence and positions of corresponding T-DNA insertions were confirmed by PCR using gene-specific and T-DNA specific primers and by sequencing the PCR products (Supplementary Table S2). With exception of line GK-175D11 (intron-insertion in Nse4B), all the other T-DNA insertions were found in exons.

Table 1

Characterization of the T-DNA insertion mutants of the A. thaliana Nse4 genes.

Gene symbolT-DNA mutantZygosityHabitPollen fertility (%)Silique length (mm)Seeds per siliqueShriveled seeds per silique% mitotic cells with bridges/fragments% meiocytes with bridges/fragments
Metaphase IAnaphase IAnaphase II
Col-wtwt100 (10790)12.8 (28)44.5 (1468)0.71.5 (417)0 (60)1.2 (67)0 (23)
Nse4ASalk_057130HeSmaller98.2 (3808)11.3 ∗∗ (24)30.3 ∗∗ (726)2.7 ∗∗ 11.9 ∗∗ (242)8.4 ∗ (59)4.2 (47)10.0 (21)
GK-768H08HoSmaller50.2 ∗∗ (6396)10.0 ∗∗ (25)21.0 ∗∗ (525)7.8 ∗∗ 25.7 ∗∗ (175)25.2 ∗∗ (115)40.4 ∗∗ (114)47.4 ∗∗ (19)
GK-768H08 (complemented)Howt-like102 (6270)12.3 ∗ (25)31.8 ∗∗ (795)3.4 ∗∗ 2.6 (373)5.0 ∗∗ (121)19.1 ∗∗ (131)15.0 ∗ (20)
Nse4BSAIL_296_F02Howt-like97.2 (3770)10.8 ∗∗ (30)30.5 ∗∗ (916)1.62.1 (278)0 (59)5.9 (85)8.3 (12)
GK-175D11Howt-like64.3 ∗∗ (3206)11.3 ∗∗ (30)34.8 ∗∗ (1080)2.9 ∗∗ 1.7 (178)6.2 ∗∗ (113)17.8 ∗∗ (106)0 (18)
Nse4A/Nse4BGK-768H08/SAIL_294_F02Ho/hoSmaller34.8 ∗∗ (4529)10.2 ∗∗ (30)17.9 ∗∗ (536)5.6 ∗∗ 28.6 ∗∗ (619)30.6 ∗∗ (72)65.0 ∗∗ (172)50.0 ∗∗ (34)

For the Nse4A lines Salk_057130 and SAIL_71_A08 only heterozygous mutants could be selected and the progeny segregated into heterozygous and wild-type plants. This indicates the requirement of Nse4A for plant viability. The confirmed truncated transcripts downstream outside of the conserved region of the homozygous line GK-768H08 ( Figure 1 and Supplementary Figure S9) obviously are able to code at least partially functional proteins. For Nse4B two homozygous lines, SAIL_296_F02 and GK-175D11, containing the T-DNA insertion in the second exon and fourth intron, respectively, were identified.

The selected mutants showed a wild-type growth habit, with only a slightly reduced plant size (especially line GK-768H08) compared to wild-type ( Figure 2A and Table 1 ). To combine the mutation effects of nse4A and nse4B, lines GK-768H08 and SAIL_296_F02 were crossed. The resulting homozygous double mutants showed a further decreased growth. The complementation of the mutation in line GK-768H08 by the genomic wild-type Nse4A construct recovered the plant viability.

Thus, the essential character of Nse4A becomes confirmed. Although knocking out of Nse4B does not induce obvious growth effects, this second Nse4 homolog is likely not completely free of function.

The selected T-DNA insertion lines were further analyzed more in detail to investigate the influence of the NSE4 proteins on meiosis and fertility. In addition to the reduced plant size, reduced pollen grain number, silique size and seed set together with shriveled seeds were observed in the mutants ( Table 1 , Figure 2B – D , and Supplementary Figure S10). The aborted seeds might represent the segregating homozygous progeny. The complementation of the mutation in line GK-768H08 by the genomic Nse4A construct recovered pollen fertility and seed setting.

To investigate the DNA damage response of the nse4 mutants compared to wild-type we applied bleomycin at different concentrations in liquid medium to induce DSBs. The treatment clearly impaired the seedling growth of both, the wild-type (Col-0) and the nse4A and nse4B mutants with increasing bleomycin concentration (Supplementary Figure S11A). To figure out whether the nse4 mutations influence the repair capacity of the plantlets, we performed a similar experiment on solid agar medium plates, and measured the seedling root lengths within 18 days growth (Supplementary Figure S11B). According to a two-way ANOVA a highly significant difference between wild-type and all mutants has been proven regarding the root development without bleomycin treatment. In addition, significantly decreased root growth rates of all three mutants were present after bleomycin application at all concentrations (0.25 0.5 and 1.0 μg/ml) (Supplementary Figure S11C). These results suggest the involvement of NSE4A and NSE4B in the repair of induced DSBs, and that their mutations may reduce the repair efficiency compared to the wild-type proteins.

NSE4 Is Essential for Correct Meiosis

The reduced number of pollen grains of the nse4 mutants suggests meiotic disturbances. Therefore, we stained meiocytes by DAPI. During prophase I no apparent alterations were found in the nse4A mutant GK-768H08 compared to wild-type. However, anaphase bridges, chromosome fragments and micronuclei appear in later meiotic stages and in tetrad cells, respectively ( Figure 3A and Supplementary Figure S12). Micronuclei are a possible product of chromosome fragmentation. In addition to line GK-768H08, all investigated nse4 mutants showed an increase in meiotic defects, with a clearly increased level in the homozygous GK-768H08/SAIL_294_F02 double mutants. The complementation of the mutation in line GK-768H08 by the genomic Nse4A construct abolished mainly the accumulation of meiotic abnormalities ( Table 1 and Supplementary Figure S13).

Meiotic defects in the nse4 mutant GK-768H08. (A) Disturbed meiosis (anaphase bridges, fragments) in the A. thaliana mutant GK-768H08 compared to wild-type (wt). (B) Chromosome fragmentation in GK-768H08 during anaphase I. Telomeres and centromeres were labeled by FISH using centromere- and telomere-specific probes. (C) Total number of subtelomeric, pericentromeric, and interstitial chromosome fragments in 18 meiotic cells of the GK-768H08 mutant. Bars = 10 μm.

To study the meiotic abnormalities more in detail, FISH experiments using 5S and 45S rDNA probes for chromosome identification were performed (Supplementary Figure S14). The analysis of the nse4A mutant GK-768H08 suggests that the occurrence of stretched bivalents, possibly causing chromosome fragments, is not related to specific chromosomes. This indicates that the defects may be induced by disturbing a general meiotic process.

Telomere- and centromere-specific FISH probes were applied to evaluate the proportion of pericentromeric, interstitial and subtelomeric fragments during anaphase I. Most fragments were found to be of subtelomeric origin, followed by interstitial fragments ( Figure 3B,C ). Obviously, the fragments are the result of a disturbed degree of chromatin condensation along rod bivalents. The increased number of rod bivalents in the mutants seems to be the consequence of a reduced recombination leading to less chiasmata. To test this hypothesis, the chiasma frequency of the nse4A mutant GK-768H08 (n = 43) was evaluated, and was found to be nearly identical with �.0 chiasmata per diakinesis/metaphase I cell to that of wild-type (Higgins et al., 2004). Thus, the truncation of NSE4A seems not to influence the number of chiasmata.

The occurrence of disturbed meiosis suggests the involvement NSE4 in meiotic processes. Indeed, transgenic A. thaliana meiocytes expressing the gNse4A::GFP construct under control of the endogenous promoter showed line-like signals at pachytene, typical for the synaptonemal complex ( Figure 4A ). In addition, by applying anti-GFP antibodies NSE4A was proven to be present in G2, leptotene, zygotene, and pachytene cells. After mainly disappearing from meta- and anaphase I chromosomes NSE4A recovered in prophase II, tetrads and young pollen ( Figure 4B ). To confirm the presence of NSE4A in a related species, immunolabeling of B. rapa meiocytes with NSE4A-specific antibodies and with ZYP1, the A. thaliana transverse filament protein of the synaptonemal complex at pachytene, was performed. The co-localization of both proteins indicated the presence of NSE4A at the synaptonemal complex during pachytene ( Figure 4C ). The immunolabeling of ZYP1 in pachytene meiocytes of the nse4A mutant GK-768H08 indicated that this mutation does not alter the synaptonemal complex structure (Supplementary Figure S15).

Localization of NSE4A during the meiosis of A. thaliana (A,B) and the closely related species B. rapa (C). (A) Line-like NSE4A-GFP signals are detectable in an unfixed meiocyte at pachytene of a transgenic pnse4A::gNse4A::GFP A. thaliana plant. (B) Dynamics and localization of NSE4A-GFP signals during meiosis of pnse4A::gNse4A::GFP transgenic A. thaliana plants, detected by anti-GFP. The NSE4A-GFP signals are detectable in G2, leptotene, zygotene, and pachytene cells. The signals are weak or not visible in condensed metaphase I and anaphase I chromosomes, respectively, but are recovered in prophase II, tetrads and young pollen. (C) Anti-AtNSE4A labels the synaptonemal complex of B. rapa and colocalizes to ZYP1 during pachytene. Gray color indicates chromatin counterstained with DAPI. Bars = 10 μm.

We conclude that both NSE4 proteins, but NSE4A again more substantially than NSE4B, are involved in meiotic processes to achieve normal fertility. However, both proteins seem not to influence the frequency of chiasmata, although NSE4A was proven to be present at the synaptonemal complex during prophase I.

NSE4 Is Present in Interphase Nuclei of Meristem and Differentiated Cells

Similar as during meiosis, abnormalities occur during mitosis in somatic flower bud nuclei of the A. thaliana nse4 mutants. These mitotic defects occur predominantly in the nse4A mutants, and less prominent in the Nse4B knock-out mutants ( Figure 5 ).

Mitotic defects (anaphase bridges, laggards) in somatic flower bud nuclei of the A. thaliana nse4 mutants (A). The diagram (B) indicates the frequency (%) of abnormalities in the mutants compared to wild-type. The percentage of abnormalities is clearly increased in the nse4A mutants SALK_057130 and GK-768H08, as well as in the homozygous double mutant GK-768H08/SAIL_296_F02 representing both nse4A and nse4B mutations, respectively. The complementation of the mutation in GK-768H08 clearly decreases the number of abnormalities indicating that they are induced by the dysfunction of the Nse4A gene. The numbers of cells analyzed are indicated above the diagram bars.

For live imaging gNSE4A::GFP signals were detected by confocal microscopy in root meristem cells. NSE4A was present in interphase nuclei, disappeared mainly during mitosis from the chromosomes and recovered at telophase at chromatin. Only a slight cytoplasm labeling remained during meta- and anaphase ( Figure 6A ). To analyze the distribution of NSE4A at the ultrastructural level, fixed interphase nuclei were stained with anti-GFP, and super-resolution microscopy (3D-SIM) has been performed. Thereby, it became obvious that NSE4A is distributed within euchromatin, but absent from nucleoli and chromocenters. During meta- and anaphase only few NSE4A signals were present within cytoplasm, confirming the live cell investigations ( Figure 6B ).

The localization of NSE4A in root meristem cells. (A) Global view of a living A. thaliana root meristem expressing a genomic NseA::GFP construct under the control of the endogenous Nse4A promoter. The cell undergoing mitosis (in the rectangle) shows that the nuclear NSE4A-GFP signals are present in interphase (0 min), disappear from the chromosomes during metaphase (2� min) and are recovered in telophase at chromatin (26 min). During metaphase a slight cytoplasm labeling is visible. (B) The ultrastructural analysis by super-resolution microscopy (SIM) confirms the presence of NSE4A within euchromatin, and indicates its absence from the nucleolus (n) and heterochromatin (chromocenters, arrows) in root meristem G1 and G2 nuclei. During meta- and anaphase NSE4A mainly disappears from the chromosomes, but stays slightly present within the cytoplasm. In young daughter nuclei (G1 phase) NSA4A becomes recovered. The localization of NSE4A-GFP expressed by pnse4A::gNse4A::GFP transgenic A. thaliana plants was detected by anti-GFP antibodies in fixed roots.

3D-SIM has also been applied to demonstrate the distribution of NSE4A in differentiated nuclei. Similar as in meristematic tissue, somatic flower bud and 8C leaf interphase nuclei display NSE4A exclusively within euchromatin ( Figure 7 ).

The distribution of NSE4A in differentiated somatic flower bud and 8C leaf interphase nuclei analyzed by 3D-SIM. In agreement, both NSE4A antibodies (anti-NSE4A) and NSE4A-EYFP signals detected by anti-GFP antibodies indicate that NSE4A is distributed within euchromatin, but absent from heterochromatin (DAPI-intense chromocenters, arrows). The NSE4A labeling visible in the merged image of the 8C nucleolus (maximum intensity projection) originates from optical sections outside of the nucleolus.

We conclude that, in addition to their meiotic function, NSE4 proteins play also a role in somatic tissue, due to its exclusive presence within the euchromatin of cycling and differentiated interphase nuclei. NSE4A is more prominent than NSE4B also in somatic tissue.

3 Design of Optogenetic Proteins

Protein engineering strategies for optogenetic proteins involve a photosensory domain (e.g., the ones previously discussed) as well as an actuating module. Their covalent connection is usually mediated with protein linkers that need to be optimized in length and structure, depending on the engineering strategy and the requirements for coupling between the modules. [ 101-103 ] The choice of the photosensory domain can be based on structural considerations of the protein itself or unique properties that different photosensory domains contain.

Such structural considerations can involve the homology to sensory domains that are naturally linked to the actuating module, which make protein engineering easier through “domain swapping” of, for example, a small molecule or hormone sensing domain with a photoactivatable domain. In other cases, the actuation module might be incorporated into the structure of a photosensory protein, which sets special structural and sequence requirements. Also, it may be of interest to reduce the size of a photosensory domain, or in other cases, to provoke sterical hindering. Similarly, certain properties of photosensory domains might be crucial for specific applications. For example, if highly dynamic optoproteins are desired, fast koff rates or light sensors that have an inactivating wavelength should be considered. However, if light-toxicity or light-delivery is problematic, high light-sensitivity and slow dark-reversion might be preferable. Also dark to light state fold-inductions of different photoregulators can be important for certain applications, although such a comparison might be case-specific and cannot always be taken from other studies in the literature. Availability of the chromophore in the used organism or medium could be another consideration.

Usually, the choice of the actuating module is very case-specific and depends on the application that the optoprotein needs to fulfill and the output it should produce, and will therefore not be further discussed. However, this section will extract general principles from successful previous optoprotein engineering efforts. Another important aspect is the screening of the functionality of optoproteins: As the protein engineering efforts might require large libraries, high-throughput methods [ 104 ] for functional screening, such as selection systems or fluorescence readouts, are preferred.

A general aspect of optogenetic proteins is that light-regulation is always based on conformational changes of the photosensory domain. Different classifications for photoactivatable proteins were previously used (e.g., [ 99, 105-108 ] ), however, we chose to classify light-regulated proteins slightly differently: To control the activity of the protein of interest, two general concepts were applied, which are either proximity- or protein-structure-based, and in some cases combined approaches are needed.

3.1 Proximity-Based Activity Control: Intermolecular Light-Control

The distance between proteins is an ubiquitous regulation factor in biology. [ 109 ] For example, assembly of multiple subcomponents is required to initiate transcription at the location of a promoter, and several factors are required to interact for protein transport or degradation. Both soluble and membrane-bound proteins are mostly symmetrical oligomeric complexes with two or more subunits. This makes complexes more stable due to reduced solvent area, provides a form of error control in protein synthesis and regulation, and allows cooperative function such as allosteric regulation and multivalent binding. [ 110 ] Chemicals and external signals can induce oligomerization of proteins, such as receptors, that subsequently initiate signaling and cellular responses. [ 111 ] The underlying regulation mechanism is based on proximity and distance of specific proteins. [ 109 ] Such proximity-based regulation is also the basis of many natural light-sensing modules in which the interaction, and therefore the distance of the interaction partners, is controlled through light. Due to allosteric changes of the photosensitive proteins upon light-induction, an interaction surface is exposed, which either allows for interaction with other identical photosensors (homodimerization or oligomerization), or for interaction with other proteins (heterodimerization). Both concepts can be utilized for proximity-based control. Therefore, proximity-based light control involves at least two fusion proteins.

Activation: inactive protein domains fused to photoactivatable domains which upon a light-input are assembled into an active protein

Recruitment: protein-recruitment of an active protein to a specific location of action

3.1.1 Activation

For the first case, light regulates assembly or release of domains necessary for the function of the protein. Prominent targets for proximity-based protein activation are signaling kinases. These kinases react to a plethora of environmental and cellular cues and control diverse functions of proteins from changing their expression, activity or localization, through altering their phosphorylation with serine, threonine, and tyrosine as their main targets. Such kinases usually show multidomain structures, containing a sensing domain that reacts to external cues often through induced oligomerization and autoactivation. [ 111 ] Light-controlled homodimerization domains were used instead of the sensing domains to implement optogenetic control usually through homodimerization. [ 112-118 ]

Heterodimerization, however, can be used if two different interaction partners are needed for the function of a protein. This is for example the case for synthetic split proteins which hold great potential for implementation of optogenetic regulation. In split protein approaches, a functional enzyme is artificially fragmented into inactive subunits. These inactive subunits are fused to light-inducible dimerization domains which reconstitute the enzyme to the active protein. [ 108, 119-121 ] An example for this is the light-inducible T7 RNA polymerase, [ 120 ] which consists of two inactive split parts that are reconstituted upon light-induced dimerization of photosensory domains (Figure 4A left).

3.1.2 Recruitment

In the second case of proximity-based control, light initiates the subcellular localization of a constitutive active protein to a location of action. For example, light-inducible heterodimerization domains were used to recruit transcription activation domains to specific sites of a promoter through promoter-bound DNA-binding proteins to initiate transcription (Figure 4A right). [ 2, 13, 122 ] In a highly similar strategy, DNA and histone modification enzymes such as DNA methyltransferases, histone deacetylases, methyltransferases, and acetyl–transferase inhibitors were used instead of transcription regulators for epigenetic regulation. [ 123, 124 ] Another widely used example is light-induced recruitment of proteins to membranes [ 125 ] where they exhibit their function, such as cAMP-dependent protein kinases which phosphorylate membrane bound substrates. [ 125, 126 ]

In both proximity-based activation and recruitment, the photosensitive domain and the effector domain(s) function independently. Engineering of such regulators mainly requires structural considerations to avoid interfering with the function of the actuator domain and to allow for correct assembly of split proteins [ 120 ] —factors that have to be considered in the choice of the light-inducible domain as well as the type of linker, its length, and its structure. [ 65, 102, 120 ]

3.2 Protein-Conformation-Based-Light Control: Intramolecular Light-Control

While proximity-based light control always involves two separate fusion proteins, allosteric conformation-based approaches only involve one protein which consists of a fusion of the effector domain and the light-inducible domain(s). The transition from the dark- to the light-induced state of photoactivatable proteins initiates a structural rearrangement that is transmitted to the effector domain leading to allosteric protein activity change or steric effects, for example, blocking and release of the active site of the effector domain.

The photoreceptors LOV2 from Avena sativa or from Arabidopsis thaliana are two prominent examples that have been used for such intramolecular light-control. The structural rearrangement upon blue-light stimulation leads to the release of a C-terminal helix (Jα-helix) from the PAS core. [ 127 ] Although this mechanism of LOV2 was also utilized for intermolecular light control, [ 128, 129 ] the dramatic conformational change makes this photosensor attractive for intramolecular light-control approaches.

3.2.1 Allosteric Regulation

Building upon studies that show that domain insertion into enzymes can be used for allosteric regulation, [ 130 ] in a pioneering example, LOV domains were used to implement light-control in a similar manner. [ 131 ] The light-induced conformational change of LOV2 is transmitted to the protein into which it is inserted, changing its activity and/or function. Such an engineering approach requires X-ray or NMR protein structures or homology models and detailed structure–function information to preselect candidate sites for insertion positions, which are usually located in surface exposed flexible loops at not conserved residues of the target protein. [ 132 ] An example are OptoNBs, [ 133 ] in which light-induced conformational changes of photosensory domains are transmitted to nanobodies leading to changes in their binding affinities (Figure 4B left).

3.2.2 Steric Regulation

In contrast to allosteric regulation, steric approaches utilize the light-induced conformational changes of a photosensory domain to change the accessibility of functionally important sites of the regulated protein (e.g., active site, regulator binding, or recognition sites). For example, the Jα-helix of LOV2 was successfully modified to incorporate signal peptides of different functions, which upon light-induction are released from the PAS core and only then fulfil their function, for example, nuclear localization, [ 134 ] protein degradation, [ 135 ] or other functional domains [ 136 ] (Figure 4B right).

Although LOV2 is a prominent example for protein conformation based light control, other photoregulators were also used for this type of control. For example Dronpa mutants were employed for light-inducible steric hindering of active sites. [ 137, 138 ] These mutants are dimeric or tetrameric, monomerize with cyan light and revert by either homodimerizing (Dronpa145K/N) or homotetramerizing (Dronpa145N) with UV light. [ 139 ] N- and C-terminal fusion of Dronpa to a protease was used for steric blockage of interaction sites, which is removed with the light-input and monomerization of the photosensor. [ 139 ]

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins*

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys 32 ) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

The atomic coordinates and structure factors (codes 3RHB and 3RHC) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (

This work was supported, in whole or in part, by National Institutes of Health Grant GM62524 (to M. K. J.). This work was also supported by the Alexander von Humboldt Foundation (to J. P. J.), Agence Nationale de la Recherche Grant JC07_204825 (to J. C. and N. R.), and Deutsche Forschungsgemeinschaft Grant Di346-14 (to K. J. D.).


I thank Matthew W. Hahn for suggestions on CAFE analysis Tomislav Domazet-Lošo for suggestions on phylostratigraphy analysis Manyuan Long, Yong Zhang, Jie Guo, and anonymous reviewers for comments about this work and Tina Hu for proofreading and improving the manuscript. Especially, I am very grateful to Detlef Weigel and Song Ge for their long-term support and also valuable comments on this work. This work was supported by 100 talents program of Chinese Academy of Sciences and the Max Planck Society.

Table S1. GO annotation of 41 rapidly evolved gene families.

Table S2. Functional annotation of 41 rapidly evolved gene families.

Table S3. Fixation rate of A. thaliana genes or gene families in each phylostratum (genes/duration of interval or gene families/duration of interval).

Table S4. The fraction of A. thaliana genes in each phylostratum that can be annotated to GO biological process.

Table S5. GO annotation of A. thaliana genes in each phylostratum in terms of proportion of major biological processes (%).

Table S6. Protein sizes of A. thaliana genes in each phylostratum.

Table S7. Expression of A. thaliana genes in each phylostratum.

Table S8. Divergences between A. thaliana genes of each phylostratum and their orthologous genes in A. lyrata.

Table S9. Divergence time of each node on the phylogeny of green plants.

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AINTEGUMENTA-like (AIL) proteins are members of the APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain family of transcription factors involved in plant growth, development, and abiotic stress responses. However, the biological functions of AIL members in pumpkin (Cucurbita moschata Duch.) remain unknown. In this study, we identified 12 AIL genes in the pumpkin genome encoding proteins predicted to be localized in the nucleus. Phylogenetic analysis showed that the AIL gene family could be classified into six major subfamilies, with each member encoding two AP2/ERF domains separated by a linker region. CmoAIL genes were expressed at varying levels in the examined tissues, and CmoANT genes showed different expression patterns under auxin (IAA), 1-naphthylphthalamic acid (NPA), and abscisic acid (ABA) treatments. Ectopic overexpression of CmoANT1.2 in Arabidopsis increased organ size and promoted growth of grafted plants by accelerating graft union formation. However, there was no significant difference at the graft junction for WT/WT and WT/ANT under IAA or NPA treatments. Taken together, the results of this study provide critical information about CmoAIL genes and their encoded proteins, and suggest future work should investigate the functions of CmoANT1.2 in the grafting process in pumpkin.

Genome-Wide Identification Analysis of the Auxin Response Factors Family in Nicotiana tabacum and the function of NtARF10 in Leaf Size Regulation

Auxin is well recognized for its involvement in several developmental processes like floral and leaf development and shoot elongation. The transcriptional regulation of auxin-responsive genes is mediated via auxin response factors (ARF). In this study, we identified 46 ARF genes in Nicotiana tabacum, performed phylogenetic analysis and investigated their structure, conserved domains, and motifs. Our results demonstrate that some of NtARF genes are regulated by mi-RNAs and expression in multiple tobacco tissues. Additionally, the leaf NtARFs display a diverse expression pattern in vein and shoot apical meristem in response to exogenous auxin stimulus. Transgenic NtARF10-overexpressing Arabidopsis plants exhibit larger leave areas, cell area, and more numerous cell numbers compared to wild-type plants and include several upregulated genes involved in cell division and expansion, including AtCYCD3, AtTCP1, AtTCP20, AtXTH33, and AtARGOS. This suggests NtARF10 might play a role in the regulation of leaf size. Our study contributes to a better understanding of the characteristics of the ARF family in tobacco and provides a basis for further functional research into NtARFs.

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We gratefully acknowledge Annette Thelen and the Genomic Technology Support Facility at Michigan State University for valuable assistance with the Affymetrix microarray analysis, Dr Thomas Whittam (Department of Microbiology, Michigan State University) and Dr Arlette Darfeuille-Michaud (Pathogenie Bacterienne Intestinale, Clermont-Ferrand, France) for providing E. coli strains, and Dr Thomas Whittam for providing lab space for work with human pathogenic bacteria. Arabidopsis EST clones used for RNA blot hybridization were obtained from the Arabidopsis Biological Resource Center. This work was supported by research grants from the US Department of Energy (DE-FG02-91ER20021) and National Institutes of Health (1R21AI060761-01) to S.Y.H., a postdoctoral fellowship to R.T. from the US Department of Agriculture, a US Department of Education GAANN Graduate Fellowship and a Michigan State University Plant Science Graduate Fellowship to W.U., and funds from the Center of Microbial Pathogenesis at Michigan State University to S. Y. H. and Thomas Whittam.

Figure S1. RNA blot analysis of differentially expressed genes identified by microarray analysis.

Figure S2. RNA blot analysis of the PAMP-responsive gene Flagellin-Induced Receptor Kinase 1 (FRK1).

Figure S3. In planta multiplication of bacterial flagellin mutants.

Figure S4. Mapman 'secondary metabolism' and 'photosynthesis' displays created using COR toxin- and TTSS-regulated genes.

Figure S5. Expression profile of genes involved in tryptophan, glucosinolate, and related biosynthetic pathways.

Figure S6. Expression profile of 44 auxin-related genes.

Figure S7. Expression profile of cytokinin-related genes.

Table S1 Non-redundant list of 2800 differentially-regulated Arabidopsis genes identified by microarray analysis

Table S2 736 PAMP-regulated Arabidopsis genes

Table S3 correlation values for the 1 × 10 8 and 5 × 10 7 bacteria mL -1 comparisons

Table S4 Known and predicted kinases induced by PAMP perception

Table S5 275 TTSS-induced Arabidopsis genes identified from the Pst COR - vs. Pst COR - hrpS comparison (5 × 10 7 bacteria mL -1 , 10 HPI)

Table S6 COR toxin-regulated Arabidopsis genes

Table S7 TTSS-regulated Arabidopsis genes

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