Genome-wide exploration of the GDSL-type esterase/lipase gene family in rapeseed reveals several BnGELP proteins active during early seedling development.

The Gly-Asp-Ser-Leu (GDSL)-type esterase/lipase proteins (GELP) are one of the most important families of lipolytic enzymes and play prominent roles in seed germination and early seedling establishment through mobilizing the lipids stored in seeds. However, there are no comprehensive studies systematically investigating the GELP gene family in Brassica napus (BnGELP), and their biological significance to these physiological processes are far from understood. In the present study, a total of 240 BnGELP genes were identified in B. napus cultivar "Zhongshuang 11" (ZS11), which is nearly 2.3-fold more GELP genes than in Arabidopsis thaliana. The BnGELP genes clustered into 5 clades based on phylogenetic analysis. Ten BnGELPs were identified through zymogram analysis of esterase activity followed by mass spectrometry, among which five clustered into the clade 5. Gene and protein architecture, gene expression, and cis-element analyses of BnGELP genes in clade 5 suggested that they may play different roles in different tissues and in response to different abiotic stresses. BnGELP99 and BnGELP159 were slightly induced by cold, which may be attributed to two low-temperature responsive cis-acting regulatory elements present in their promoters. An increased activity of esterase isozymes by cold was also observed, which may reflect other cold inducible esterases/lipases in addition to the ten identified BnGELPs. This study provides a systemic view of the BnGELP gene family and offers a strategy for researchers to identify candidate esterase/lipase genes responsible for lipid mobilization during seed germination and early seedling establishment.

Genome-Wide Analysis of MYB Transcription Factor Gene Superfamily Reveals BjPHL2a Involved in Modulating the Expression of BjCHI1 in Brassica juncea.

Brassica juncea is an economically important vegetable and oilseed crop. The MYB transcription factor superfamily is one of the largest transcription factor families in plants, and plays crucial roles in regulating the expression of key genes involved in a variety of physiological processes. However, a systematic analysis of the MYB transcription factor genes in Brassica juncea (BjMYB) has not been performed. In this study, a total of 502 BjMYB superfamily transcription factor genes were identified, including 23 1R-MYBs, 388 R2R3-MYBs, 16 3R-MYBs, 4 4R-MYBs, 7 atypical MYBs, and 64 MYB-CCs, which is approximately 2.4-fold larger than that of AtMYBs. Phylogenetic relationship analysis revealed that the MYB-CC subfamily consists of 64 BjMYB-CC genes. The expression pattern of members of PHL2 subclade homologous genes in Brassica juncea (BjPHL2) after Botrytis cinerea infection were determined, and BjPHL2a was isolated from a yeast one-hybrid screen with the promoter of BjCHI1 as bait. BjPHL2a was found to localize mainly in the nucleus of plant cells. An EMSA assay confirmed that BjPHL2a binds to the Wbl-4 element of BjCHI1. Transiently expressed BjPHL2a activates expression of the GUS reporter system driven by a BjCHI1 mini-promoter in tobacco (Nicotiana benthamiana) leaves. Taken together, our data provide a comprehensive evaluation of BjMYBs and show that BjPHL2a, one of the members of BjMYB-CCs, functions as a transcription activator by interacting with the Wbl-4 element in the promoter of BjCHI1 for targeted gene-inducible expression.

Molecular identification of BrHAB2a, one of the two AtHAB2-like proteins in Brassica rapa, is an important component of ABA signaling.

Abscisic acid (ABA) signaling is a vital physiological step that is used by many land plants to fight against environmental stress. As components of the linear ABA signaling pathway, clade A protein phosphatases type 2C (PP2C-As) are mainly inhibited by PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors upon their binding to ABA. Here, we show that the genome of Brassica rapa encodes 14 putative clade A PP2C-like proteins (BrPP2C-As). Two of these BrPP2C-As, Bra025964 and Bra016595, show high similarity to the HAB2 (Homology to ABI2) protein in Arabidopsis. RNAseq data reveal that nearly all BrPP2C-As, like BrHAB2a (Bra025964) and BrHAB2b (Bra016595), were highly expressed in at least one tissue. Overexpression of BrHAB2a conferred ABA insensitivity to Arabidopsis thaliana seedlings. Furthermore, the phosphatase activity of BrHAB2a could be inhibited by AtPYL1 or BrPYL1 in the presence of ABA. Overall, these results suggest that BrHAB2a is a functional PP2C-A like protein phosphotase and a key component of ABA signaling in Brassica rapa.

Molecular Characterization, Gene Evolution, and Expression Analysis of the Fructose-1, 6-bisphosphate Aldolase (FBA) Gene Family in Wheat (Triticum aestivum L.).

Fructose-1, 6-bisphosphate aldolase (FBA) is a key plant enzyme that is involved in glycolysis, gluconeogenesis, and the Calvin cycle. It plays significant roles in biotic and abiotic stress responses, as well as in regulating growth and development processes. In the present paper, 21 genes encoding TaFBA isoenzymes were identified, characterized, and categorized into three groups: class I chloroplast/plastid FBA (CpFBA), class I cytosol FBA (cFBA), and class II chloroplast/plastid FBA. By using a prediction online database and genomic PCR analysis of Chinese Spring nulli-tetrasomic lines, we have confirmed the chromosomal location of these genes in 12 chromosomes of four homologous groups. Sequence and genomic structure analysis revealed the high identity of the allelic TaFBA genes and the origin of different TaFBA genes. Numerous putative environment stimulus-responsive cis-elements have been identified in 1,500-bp regions of TaFBA gene promoters, of which the most abundant are the light-regulated elements (LREs). Phylogenetic reconstruction using the deduced protein sequence of 245 FBA genes indicated an independent evolutionary pathway for the class I and class II groups. Although, earlier studies have indicated that class II FBA only occurs in prokaryote and fungi, our results have demonstrated that a few class II CpFBAs exist in wheat and other closely related species. Class I TaFBA was predicted to be tetramers and class II to be dimers. Gene expression analysis based on microarray and transcriptome databases suggested the distinct role of TaFBAs in different tissues and developmental stages. The TaFBA 4-9 genes were highly expressed in leaves and might play important roles in wheat development. The differential expression patterns of the TaFBA genes in light/dark and a few abiotic stress conditions were also analyzed. The results suggested that LRE cis-elements of TaFBA gene promoters were not directly related to light responses. Most TaFBA genes had higher expression levels in the roots than in the shoots when under various stresses. Class I cytosol TaFBA genes, particularly TaFBA10/12/18 and TaFBA13/16, and three class II TaFBA genes are involved in responses to various abiotic stresses. Class I CpFBA genes in wheat are apparently sensitive to different stress conditions.

Molecular characterization of an AtPYL1-like protein, BrPYL1, as a putative ABA receptor in Brassica rapa.

Abscisic acid (ABA)-induced physiological changes are conserved in many land plants and underlie their responses to environmental stress and pathogens. The PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors perceive the ABA signal and initiate signal transduction. Here, we show that the genome of Brassica rapa encodes 24 putative AtPYL-like proteins. The AtPYL-like proteins in Brassica rapa (BrPYLs) can also be classified into 3 subclasses. We found that nearly all BrPYLs displayed high expression in at least one tissue. Overexpression of BrPYL1 conferred ABA hypersensitivity to Arabidopsis. Further, ABA activated the expression of an ABA-responsive reporter in Arabidopsis protoplasts expressing BrPYL1. Overall, these results suggest that BrPYL1 is a putative functional ABA receptor in Brassica rapa.

Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage.

bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.

SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type Protein Kinase, Is Important for Abscisic Acid Responses in Arabidopsis through Phosphorylation of ABSCISIC ACID-INSENSITIVE5.

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-related protein kinase3-type protein kinase, SOS2-like protein kinase5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, abscisic acid-insensitive5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-like calcium binding proteins) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana).

BolOST1, an ortholog of Open Stomata 1 with alternative splicing products in Brassica oleracea, positively modulates drought responses in plants.

Open Stomata 1 (OST1), an ABA-activated sucrose non-fermenting 1 (SNF1)-related protein kinase, is critical for plant drought responses. We investigated the functions of two splicing isoforms of the OST1 ortholog in Brassica oleracea (BolOST1). BolOST1 expression was found to be dramatically induced by drought and high-salt stress, and the ectopic expression of BolOST1 restored the drought-sensitive phenotype of ost1. Subcellular localization revealed that BolOST1 is localized in both the nucleus and cytoplasm. BolOST1 was also demonstrated to phosphorylate the N-terminal fragment of ABI5 (ABA Insensitive 5, ABI5-N). A firefly luciferase complementation assay revealed that BolOST1 interacts with both BolABI5 and an ABI1 ortholog in B. oleracea (BolABI1). Overall, these results suggest that BolOST1 is a functional SnRK2-type protein kinase and that the early ABA signaling network may be conserved between Arabidopsis and cabbage.

Negative regulation of abscisic acid signaling by the Brassica oleracea ABI1 ortholog.

ABI1 (ABA Insensitive 1) is an important component of the core regulatory network in early ABA (Abscisic acid) signaling. Here, we investigated the functions of an ABI1 ortholog in Brassica oleracea (BolABI1). The expression of BolABI1 was dramatically induced by drought, and constitutive expression of BolABI1 confers ABA insensitivity upon the wild-type. Subcellular localization and phosphatase assays reveal that BolABI1 is predominantly localized in the nucleus and harbors phosphatase activity. Furthermore, BolABI1 interacts with a homolog of OST1 (OPEN STOMATA 1) in B. oleracea (BolOST1) and can dephosphorylate ABI5 (ABA Insensitive 5) in vitro. Overall, these results suggest that BolABI1 is a functional PP2C-type protein phosphatase that is involved in the negative modulation of the ABA signaling pathway.

Molecular characterization of an ABA insensitive 5 orthologue in Brassica oleracea.

ABI5 (ABA insensitive 5), a bZIP (Basic leucine zipper) transcription factor, has been shown to be a major mediator of plant ABA responses during seed germination. Although the molecular basis of ABI5-modulated processes has been well demonstrated in Arabidopsis thaliana, its identity and function in cabbage (Brassica oleracea var. capitata L.) remain elusive. Here, we describe our identification of BolABI5 (an ABI5 orthologue in B.oleracea) as a functional bZIP transcription factor in the modulation of plant ABA responses. Expression of BolABI5 was dramatically induced by drought stress and exogenous ABA. Heterogeneous expression of BolABI5 rescued the insensitive phenotype of Arabidopsis abi5-1 to ABA during seed germination. Subcellular localization and trans-activation assays revealed that BolABI5 was localized in the nucleus and possessed DNA binding and trans-activation activities. Deletion of the bZIP domain generated BolABI5ΔbZIP, which no longer localized exclusively in the nucleus and had almost no detectable DNA-binding or trans-activation activities. Overall, these results suggest that BolABI5 may function as ABI5 in the positive regulation of plant ABA responses.

Roles of SCaBP8 in salt stress response.

The tissue-preferential distributed calcium sensors, SOS3 and SCaBP8, play important roles in SOS pathway to cope with saline conditions. Both SOS3 and SCaBP8 interact with and activate SOS2. However the regulatory mechanism for SOS2 activation and membrane recruitment by SCaBP8 differs from SOS3. SCaBP8 is phosphorylated by SOS2 at plasma membrane (PM) under salt stress. This phosphorylation anchors the SCaBP8-SOS2 complex on plasma membrane and activates PM Na(+)/H(+) antiporter, such as SOS1. Here, we describe that SOS2 has high binding affinity and catalytic efficiency to SCaBP8, suggesting that phosphorylation of SCaBP8 by SOS2 perhaps occurs rapidly in salt condition. SCaBP8 is also phosphorylated by PKS5 (SOS2-like Protein Kinase5) which negatively regulates PM H(+)-ATPase activity and functions in plant alkaline tolerance, providing a clue to roles of SCaBP8 in both salt and alkaline tolerance. SOS2 interacts with SOS3 and SCaBP8 with its FISL motif at C-terminus. However, luciferase activity complement assay indicates that SOS2 N-terminal is also essential for interacting with these proteins in plant.