Functional and regulatory diversity of homeobox-leucine zipper transcription factors BnaHB6 under dehydration and salt stress in Brassica napus L.

The plant-specific homeodomain-leucine zipper I subfamily is involved in the regulation of various biological processes, particularly growth, development and stress response. In the present study, we characterized four BnaHB6 homologues from Brassica napus. All BnaHB6 proteins have transcriptional activation activity. Structural and functional data indicate the complex role of BnaHB6 genes in regulating biological processes, with some functions conserved and others diverged. Transcriptional analyzes revealed that they are induced in a similar manner in different tissues but show different expression patterns in response to stress and circadian rhythm. Only the BnaA09HB6 and BnaC08HB6 genes are expressed under dehydration and salt stress, and in darkness. The partial transcriptional overlap of BnaHB6s with the evolutionarily related genes BnaHB5 and BnaHB16 was also observed. Transgenic Arabidopsis thaliana plants expressing a single proBnaHB6::GUS partially confirmed the expression results. Bioinformatic analysis allowed the identification of TF-binding sites in the BnaHB6 promoters that may control their expression under stress and circadian rhythm. ChIP-qPCR analysis revealed that BnaA09HB6 and BnaC08HB6 bind directly to the promoters of the target genes BnaABF4 and BnaDREB2A. Comparison of their expression patterns in the WT plants and the bnac08hb6 mutant showed that BnaC08HB6 positively regulates the expression of the BnaABF4 and BnaDREB2A genes under dehydration and salt stress. We conclude that four BnaHB6 homologues have distinct functions in response to stress despite high sequence similarity, possibly indicating different binding preferences with BnaABF4 and BnaDREB2A. We hypothesize that BnaC08HB6 and BnaA09HB6 function in a complex regulatory network under stress.

Evolutionary Consequences of Functional and Regulatory Divergence of HD-Zip I Transcription Factors as a Source of Diversity in Protein Interaction Networks in Plants.

The HD superfamily has been studied in detail for several decades. The plant-specific HD-Zip I subfamily attracts the most attention because of its involvement in plant development and stress responses. In this review, we provide a comprehensive insight into the evolutionary events responsible for the functional redundancy and diversification of the HD-Zip I genes in regulating various biological processes. We summarized the evolutionary history of the HD-Zip family, highlighting the important role of WGDs in its expansion and divergence of retained duplicates in the genome. To determine the relationship between the evolutionary origin and functional conservation of HD-Zip I in different species, we performed a phylogenetic analysis, compared their expression profiles in different tissues and under stress and traced the role of orthologs and paralogs in regulating developmental processes. We found that HD-Zip I from different species have similar gene structures with a highly conserved HD and Zip, bind to the same DNA sequences and are involved in similar biological processes. However, they exhibit a functional diversity, which is manifested in altered expression patterns. Some of them are involved in the regulation of species-specific leaf morphology and phenotypes. Here, we discuss the role of changes in functional domains involved in DNA binding and protein interaction of HD-Zip I and in cis-regulated regions of its target genes in promoting adaptive innovations through the formation of de novo regulatory systems. Understanding the role of the HD-Zip I subfamily in organism-environment interactions remains a challenge for evolutionary developmental biology (evo-devo).

Functional divergence of Brassica napus BnaABI1 paralogs in the structurally conserved PP2CA gene subfamily of Brassicaceae.

Group A PP2C (PP2CA) genes form a gene subfamily whose members play an important role in regulating many biological processes by dephosphorylation of target proteins. In this study we examined the effects of evolutionary changes responsible for functional divergence of BnaABI1 paralogs in Brassica napus against the background of the conserved PP2CA gene subfamily in Brassicaceae. We performed comprehensive phylogenetic analyses of 192 PP2CA genes in 15 species in combination with protein structure homology modeling. Fundamentally, the number of PP2CA genes remained relatively constant in these taxa, except in the Brassica genus and Camelina sativa. The expansion of this gene subfamily in these species has resulted from whole genome duplication. We demonstrated a high degree of structural conservation of the PP2CA genes, with a few minor variations between the different PP2CA groups. Furthermore, the pattern of conserved sequence motifs in the PP2CA proteins and their secondary and 3D structures revealed strong conservation of the key ion-binding sites. Syntenic analysis of triplicated regions including ABI1 paralogs revealed significant structural rearrangements of the Brassica genomes. The functional and syntenic data clearly show that triplication of BnaABI1 in B. napus has had an impact on its functions, as well as the positions of adjacent genes in the corresponding chromosomal regions. The expression profiling of BnaABI1 genes showed functional divergence, i.e. subfunctionalization, potentially leading to neofunctionalization. These differences in expression are likely due to changes in the promoters of the BnaABI1 paralogs. Our results highlight the complexity of PP2CA gene subfamily evolution in Brassicaceae.

Expression profiling of the genes encoding ABA route components and the ACC oxidase isozymes in the senescing leaves of Populus tremula.

Abscisic acid (ABA) triggers and regulates, while ethylene modulates autumn leaf senescence. The expression profiles of genes encoding ABA route components and the ACC oxidase isozymes were investigated in Populus tremula during the early and moderate stages of autumn leaf senescence. The targets of interest were Ptre-HAB1-like genes (Ptre-HAB1, Ptre-HAB3a and Ptre-HAB3b), the subclass 3 of Ptre-SnRK2s genes (Ptre-SnRK2.6a, Ptre-SnRK2.6b and Ptre-SnRK2.6b) and Ptre-RbohD1, Ptre-RbohF1, and Ptre-RbohF2 genes encoding the poplar components, which are counterparts of the ABA route key regulators or the counterparts of its secondary messengers, such as Homology to ABA-insensitive 1 (HAB1), Sucrose non-fermenting 1-related protein kinases 2 (SnRK2s) or Respiratory burst oxidase D and Respiratory burst oxidase F (RbohD and RbohF, respectively) in Arabidopsis, and Ptre-ACO3, Ptre-ACO5, and Ptre-ACO6 genes encoding ACC oxidase isozymes involved in ethylene biosynthesis. The fold change in their expression levels enabled to distinguish the distinct expression patterns for the following pairs of genes: Ptre-HAB3a and Ptre-SnRK2.6a, Ptre-HAB3b and Ptre-SnRK2.2, and Ptre-HAB1 and Ptre-SnRK2.6b, where each pair involves the genes encoding the negative and positive regulators of ABA route, respectively. Among the investigated genes, the fold change of expression was the highest for Ptre-ACO3, Ptre-ACO6, and Ptre-SnRK2.6b genes during both the studied stages, and additionally for Ptre-HAB1 and Ptre-RbohD1 genes during the moderate stage. In contrast, the Ptre-RbohF1 and Ptre-RbohF2 genes exhibited only the transient upregulation at the early stage of senescence. In an in vitro study, the ability of protein kinases Ptre-SnRK2.6a and Ptre-SnRK2.6b to phosphorylate the N-terminal regions of Ptre-RbohD1 and Ptre-RbohF2 was studied; the activity of Ptre-SnRK2.6b against the studied Ptre-Rbohs was noticeably lower than that exhibited by Ptre-SnRK2.6a. It seems that despite the high similarity of their polypeptides, Ptre-SnRK2.6a and Ptre-SnRK2.6b may play different biological roles; nonetheless, it requires in vivo confirmation. Surprisingly, the highest protein kinase activity against the Ptre-Rbohs was detected in the heterologous reaction with AT-SnRK2.6/OST1 which suggests that the discussed interactions are evolutionary conserved.

Expression profiling of CTR1-like and EIN2-like genes in buds and leaves of Populus tremula, and in vitro study of the interaction between their polypeptides.

In Arabidopsis, the serine/threonine protein kinase Constitutive Triple Response 1 (CTR1) and Ethylene Insensitive 2 polypeptide (EIN2) functions are key negative and positive components, respectively, in the ethylene signalling route. Here, we report on an in silico study of members of the CTR1-like and EIN2-like polypeptide families from poplars. The expression of CTR1-like and EIN2-like genes such as Ptre-CTR1, Ptre-CTR3 and Ptre-EIN2a was studied in Populus tremula buds and leaves in response to dehydration, various light conditions and under senescence. In buds under dehydration, the maximal fold-change of the Ptre-CTR1, Ptre-CTR3 and Ptre-EIN2a expression level recorded almost identical values. This suggests that maintenance of a constant ratio between the transcript levels of genes encoding positive and negative ethylene signalling components is required under stress. The expression of the studied genes was 1.4-to 3-fold higher in response to darkness, but 4.5- to 51.2-fold and 21.6- to 51.2-fold higher under the early and moderate leaf senescence, respectively. It is worth noting that the senescence-related Ptre-EIN2a and Ptre-CTR3a expression profiles were very similar. Using in vitro assays, we demonstrated the ability of the catalytic domain of Ptre-CTR1 to phosphorylate the Ptre-EIN2a-like polypeptide, which is similar to that in Arabidopsis. The target substrate, the Ptre-CEND2a polypeptide (C-terminal part of Ptre-EIN2a), was only phosphorylated by the protein kinase Ptre-CTR1 and not by Ptre-CTR3. Moreover, the addition of Ptre-CTR3 polypeptides (-CTR3a or -CTR3b forms) to the reaction mixture had an inhibitory effect on Ptre-CTR1 auto- and trans-phosphorylation. In contrast to Ptre-CTR1, Ptre-CTR3 may act as a positive regulator in ethylene signalling in poplar; however, this hypothesis requires in vivo confirmation. Thus, the ethylene signalling route in poplar seems to be under the control of certain additional mechanisms which have not been reported in Arabidopsis.

Expression profiling of genes encoding ABA route components in response to dehydration or various light conditions in poplar buds and leaves.

In this report, the members of PP2C, SnRK2a and Rboh oxidase families from Arabidopsis and poplar were studied in silico, and the expression profiles of the some of them were specified in Populus tremula buds and adult leaves. In poplars, the counterparts of ABI1- and ABI2-like protein phosphatases are lacking, but poplar genomes encode three HAB-like proteins denoted in this work as HAB1, HAB3a and HAB3b, and the counterparts of the two latter ones are absent in Arabidopsis. Nonetheless, they may be present in other species. In poplars, SnRK2 subclass III includes two SnRK2.6-like protein kinases denoted by us as SnRK2.6a and SnRK2.6b, and only one SnRK2.2 corresponding to SnRK2.2 and SnRK2.3 ones from Arabidopsis. In contrast to Arabidopsis, the poplar Rboh family involves two RbohD- and RbohF-like proteins denoted here as RbohD1 and RbohD2, and RbohF1 and RbohF2, respectively. The expressions of genes encoding the above components of the ABA route were studied in Populus tremula dehydrated buds and adult leaves not subjected to stress but exposed to natural daylight or to darkness, and to inhibition of ethylene biosynthesis or signaling route by cobalt or silver ions, respectively. In leaves, the light conditions seemed to be the most pronounced factor, from among the studied stimuli, controlling the expression Ptre-HAB3a, Ptre-HAB1, Ptre-SnRK2.6a and Ptre-RbohF2 genes, their expression was upregulated in darkness. This observation implies that these genes may be important for dark-induced stomatal closure regulation. Ethylene negatively affected the expression of three studied Rboh genes and Ptre-HAB1one but only at daylight, whereas its positive effect on the of Ptre-HAB3a was shown in the dark exposed leaves. In buds, three studied Rboh genes took part in the early response to dehydration, however their participation involved the visibly highest level of the Ptre-RbohD1 transcripts, followed by Ptre-RbohF2 and the lowest one of Ptre-RbohF1. Nonetheless, the further stress-induced superoxide anion generation seemed to depend on the enhanced expression of the Ptre-RbohD1 and Ptre-RbohF2 genes only, still with a significantly higher level of the Ptre-RbohD1 one. Ptre-RbohD2 transcripts were found neither in leaves nor in buds. The expression of the other genes discussed in the present work was either slightly upregulated at moderate stress or did not significantly change in response to dehydration. The protein kinase activity of overexpressed Ptre-SnRK2.6a and Ptre-SnRK2.6b was confirmed in in vitro protein kinase assay and compared to that of SnRK2.6/OST1 one from Arabidopsis.

Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin-Proteasome Pathway.

Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.

Involvement of genes encoding ABI1 protein phosphatases in the response of Brassica napus L. to drought stress.

In this report we characterized the Arabidopsis ABI1 gene orthologue and Brassica napus gene paralogues encoding protein phosphatase 2C (PP2C, group A), which is known to be a negative regulator of the ABA signaling pathway. Six homologous B. napus sequences were identified and characterized as putative PP2C group A members. To gain insight into the conservation of ABI1 function in Brassicaceae, and understand better its regulatory effects in the drought stress response, we generated transgenic B. napus plants overexpressing A. thaliana ABI1. Transgenic plants subjected to drought showed a decrease in relative water content, photosynthetic pigments content and expression level of RAB18- and RD19A-drought-responsive marker genes relative to WT plants. We present the characterization of the drought response of B. napus with the participation of ABI1-like paralogues. The expression pattern of two evolutionarily distant paralogues, BnaA01.ABI1.a and BnaC07.ABI1.b in B. napus and their promoter activity in A. thaliana showed differences in the induction of the paralogues under dehydration stress. Comparative sequence analysis of both BnaABI1 promoters showed variation in positions of cis-acting elements that are especially important for ABA- and stress-inducible expression. Together, these data reveal that subfunctionalization following gene duplication may be important in the maintenance and functional divergence of the BnaABI1 paralogues. Our results provide a framework for a better understanding of (1) the role of ABI1 as a hub protein regulator of the drought response, and (2) the differential involvement of the duplicated BnaABI1 genes in the response of B. napus to dehydration-related stresses.

Hyperspectral and thermal imaging of oilseed rape (Brassica napus) response to fungal species of the genus Alternaria.

In this paper, thermal (8-13 µm) and hyperspectral imaging in visible and near infrared (VNIR) and short wavelength infrared (SWIR) ranges were used to elaborate a method of early detection of biotic stresses caused by fungal species belonging to the genus Alternaria that were host (Alternaria alternata, Alternaria brassicae, and Alternaria brassicicola) and non-host (Alternaria dauci) pathogens to oilseed rape (Brassica napus L.). The measurements of disease severity for chosen dates after inoculation were compared to temperature distributions on infected leaves and to averaged reflectance characteristics. Statistical analysis revealed that leaf temperature distributions on particular days after inoculation and respective spectral characteristics, especially in the SWIR range (1000-2500 nm), significantly differed for the leaves inoculated with A. dauci from the other species of Alternaria as well as from leaves of non-treated plants. The significant differences in leaf temperature of the studied Alternaria species were observed in various stages of infection development. The classification experiments were performed on the hyperspectral data of the leaf surfaces to distinguish days after inoculation and Alternaria species. The second-derivative transformation of the spectral data together with back-propagation neural networks (BNNs) appeared to be the best combination for classification of days after inoculation (prediction accuracy 90.5%) and Alternaria species (prediction accuracy 80.5%).

Comparative analysis of the Brassica oleracea genetic map and the Arabidopsis thaliana genome.

We further investigated genome macrosynteny for Brassica species and Arabidopsis thaliana. This work aimed at comparative map construction for B. oleracea and A. thaliana chromosomes based on 160 known A. thaliana probes: 147 expressed sequence tags (ESTs) and 13 full-length cDNA clones. Based on an in silico study of the A. thaliana genome, most of the selected ESTs (83%) represented unique or low-copy genes. We identified conserved segments by the visual inspection of comparative data with a priori assumptions, and established their significance with the LineUp algorithm. Evaluation of the number of B. oleracea gene copies per A. thaliana EST revealed a fixed upward trend. We established a segregation distortion pattern for all genetic loci, with particular consideration of the type of selection (gametic or zygotic), and discuss its possible impact on genetic map construction. Consistent with previous reports, we found evidence for numerous chromosome rearrangements and the genome fragment replication of B. oleracea that have taken place since the divergence of the two species. Also, we found that over 54% of the B. oleracea genome is covered by 24 segments conserved with the A. thaliana genome. The average conserved segment is composed of 5 loci covering 19.3 cM in the B. oleracea genetic map and 2.42 Mb in the A. thaliana physical map. We have also attempted to use a unified system of conserved blocks (previously described) to verify our results and perform a comprehensive comparison with other Brassica species.