The plasma membrane H + -ATPase (PM H + -ATPase) plays an important role in the regulation of ion and metabolite transport and is involved in physiological processes that include cell growth, intracellular pH, and stomatal regulation. PM H + -ATPase activity is controlled by many factors, including hormones, calcium, light, and environmental stresses like increased soil salinity. We have previously shown that the Arabidopsis thaliana Salt Overly Sensitive2-Like Protein Kinase5 (PKS5) negatively regulates the PM H + -ATPase. Here, we report that a chaperone, J3 (DnaJ homolog 3; heat shock protein 40-like), activates PM H + -ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 are hypersensitive to salt at high external pH and exhibit decreased PM H + -ATPase activity. J3 functions upstream of PKS5 as double mutants generated using j3-1 and several pks5 mutant alleles with altered kinase activity have levels of PM H + -ATPase activity and responses to salt at alkaline pH similar to their corresponding pks5 mutant. Taken together, our results demonstrate that regulation of PM H + -ATPase activity by J3 takes place via inactivation of the PKS5 kinase.

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.

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The leucine-rich repeat receptor-like protein kinase (LRR-RLK) family represents the largest group of RLKs in plants and plays vital roles in plant growth, development and the responses to environmental stress. Although LRR-RLK families have been identified in many species, they have not yet been reported in B. napus. In this study, a total of 444 BnLRR-RLK genes were identified in the genome of Brassica napus cultivar “Zhongshuang 11” (ZS11), and classified into 22 subfamilies based on phylogenetic relationships and genome-wide analyses. Conserved motifs and gene structures were shared within but not between subfamilies. The 444 BnLRR-RLK genes were asymmetrically distributed on 19 chromosomes and exhibited specific expression profiles in different tissues and in response to stress. We identified six BnBRI1 homologs and obtained partial knockouts via CRISPR/Cas9 technology, generating semi-dwarf lines without decreased yield compared with controls. This study provides comprehensive insight of the LRR-RLK family in B. napus. Additionally, the semi-dwarf lines expand the “ideotype” germplasm resources and accelerate the breeding process for B. napus.

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The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar “Zhongshuang 11” (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes.