Multi-algorithm cooperation research of WRKY genes under nitrogen stress in Panax notoginseng.

WRKY transcription factors play an important role in the immune system and the innate defense response of plants. WRKY transcription factors have great feedback on nitrogen stress. In this study, bioinformatics was used to detect the WRKYs of Panax notoginseng (PnWRKYs). The response of PnWRKYs under nitrogen stress was also well studied. PnWRKYs were distributed on 11 chromosomes. According to PnWRKY and Arabidopsis thaliana WRKY (AtWRKY) domains, these PnWRKY proteins were divided into three groups by phylogenetic analysis. MEME analysis showed that almost every member contained motif 1 and motif 2. PlantCARE online predicted the cis-acting elements of the promoter. PnWRKY gene family members obtained 22 pairs of repeat fragments by collinearity analysis. The expression levels of PnWRKYs in different parts (roots, flowers, and leafs) were analyzed by the gene expression pattern. They reflected tissue-specific expressions. The qRT-PCR experiments were used to detect 74 PnWRKYs under nitrogen stress. The results showed that the expression levels of 8 PnWRKYs were significantly induced. The PnWRKY gene family may be involved in biotic/abiotic stresses and hormone induction. This study will not only lay the foundation to explore the functions of PnWRKYs but also provide candidate genes for the future improvement of P. notoginseng.

The Anaerobic Product Ethanol Promotes Autophagy-Dependent Submergence Tolerance in Arabidopsis.

In response to hypoxia under submergence, plants switch from aerobic respiration to anaerobic fermentation, which leads to the accumulation of the end product, ethanol. We previously reported that Arabidopsis thaliana autophagy-deficient mutants show increased sensitivity to ethanol treatment, indicating that ethanol is likely involved in regulating the autophagy-mediated hypoxia response. Here, using a transcriptomic analysis, we identified 3909 genes in Arabidopsis seedlings that were differentially expressed in response to ethanol treatment, including 2487 upregulated and 1422 downregulated genes. Ethanol treatment significantly upregulated genes involved in autophagy and the detoxification of reactive oxygen species. Using transgenic lines expressing AUTOPHAGY-RELATED PROTEIN 8e fused to green fluorescent protein (GFP-ATG8e), we confirmed that exogenous ethanol treatment promotes autophagosome formation in vivo. Phenotypic analysis showed that deletions in the alcohol dehydrogenase gene in adh1 mutants result in attenuated submergence tolerance, decreased accumulation of ATG proteins, and diminished submergence-induced autophagosome formation. Compared to the submergence-tolerant Arabidopsis accession Columbia (Col-0), the submergence-intolerant accession Landsberg erecta (Ler) displayed hypersensitivity to ethanol treatment; we linked these phenotypes to differences in the functions of ADH1 and the autophagy machinery between these accessions. Thus, ethanol promotes autophagy-mediated submergence tolerance in Arabidopsis.

Evolutionary conservation and post-translational control of S-adenosyl-L-homocysteine hydrolase in land plants.

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.

Functional Divergence of the Arabidopsis Florigen-Interacting bZIP Transcription Factors FD and FDP.

Flowering of many plant species depends on interactions between basic leucine zipper (bZIP) transcription factors and systemically transported florigen proteins. Members of the genus Arabidopsis contain two of these bZIPs, FD and FDP, which we show have largely complementary expression patterns in shoot apices before and during flowering. CRISPR-Cas9-induced null mutants for FDP flower slightly earlier than wild-type, whereas fd mutants are late flowering. Identical G-box sequences are enriched at FD and FDP binding sites, but only FD binds to genes involved in flowering and only fd alters their transcription. However, both proteins bind to genes involved in responses to the phytohormone abscisic acid (ABA), which controls developmental and stress responses. Many of these genes are differentially expressed in both fd and fdp mutant seedlings, which also show reduced ABA sensitivity. Thus, florigen-interacting bZIPs have distinct functions in flowering dependent on their expression patterns and, at earlier stages in development, play common roles in phytohormone signaling.

The atypical histone variant H3.15 promotes callus formation in Arabidopsis thaliana.

Plants are capable of regenerating new organs after mechanical injury. The regeneration process involves genome-wide reprogramming of transcription, which usually requires dynamic changes in the chromatin landscape. We show that the histone 3 variant HISTONE THREE RELATED 15 (H3.15) plays an important role in cell fate reprogramming during plant regeneration in Arabidopsis H3.15 expression is rapidly induced upon wounding. Ectopic overexpression of H3.15 promotes cell proliferation to form a larger callus at the wound site, whereas htr15 mutation compromises callus formation. H3.15 is distinguished from other Arabidopsis histones by the absence of the lysine residue 27 that is trimethylated by the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) in constitutively expressed H3 variants. Overexpression of H3.15 promotes the removal of the transcriptional repressive mark H3K27me3 from chromatin, which results in transcriptional de-repression of downstream genes, such as WUSCHEL RELATED HOMEOBOX 11 (WOX11). Our results reveal a new mechanism for a release from PRC2-mediated gene repression through H3.15 deposition into chromatin, which is involved in reprogramming cell fate to produce pluripotent callus cells.

A phylogenetically conserved APETALA2/ETHYLENE RESPONSE FACTOR, ERF12, regulates Arabidopsis floral development.

KEY MESSAGE: Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRÖSCHEN, DORNRÖSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity.

ZmMYC2 exhibits diverse functions and enhances JA signaling in transgenic Arabidopsis.

KEY MESSAGE: ZmMYC2 was identified as the key regulator of JA signaling in maize and exhibited diverse functions through binding to many gene promoters as well as enhanced JA signaling in transgenic Arabidopsis. The plant hormone jasmonate (JA) extensively coordinates plant growth, development and defensive responses. MYC2 is the master regulator of JA signaling and has been widely studied in many plant species. However, little is known about this transcription factor in maize. Here, we identified one maize transcription factor with amino acid identity of 47% to the well-studied Arabidopsis AtMYC2, named as ZmMYC2. Gene expression analysis demonstrated inducible expression patterns of ZmMYC2 in response to multiple plant hormone treatments, as well as biotic and abiotic stresses. The yeast two-hybrid assay indicated physical interaction among ZmMYC2 and JA signal repressors ZmJAZ14, ZmJAZ17, AtJAZ1 and AtJAZ9. ZmMYC2 overexpression in Arabidopsis myc2myc3myc4 restored the sensitivity to JA treatment, resulting in shorter root growth and inducible anthocyanin accumulation. Furthermore, overexpression of ZmMYC2 in Arabidopsis elevated resistance to Botrytis cinerea. Further ChIP-Seq analysis revealed diverse regulatory roles of ZmMYC2 in maize, especially in the signaling crosstalk between JA and auxin. Hence, we identified ZmMYC2 and characterized its roles in regulating JA-mediated growth, development and defense responses.

SH3Ps-Evolution and Diversity of a Family of Proteins Engaged in Plant Cytokinesis.

SH3P2 (At4g34660), an Arabidopsis thaliana SH3 and Bin/amphiphysin/Rvs (BAR) domain-containing protein, was reported to have a specific role in cell plate assembly, unlike its paralogs SH3P1 (At1g31440) and SH3P3 (At4g18060). SH3P family members were also predicted to interact with formins-evolutionarily conserved actin nucleators that participate in microtubule organization and in membrane-cytoskeleton interactions. To trace the origin of functional specialization of plant SH3Ps, we performed phylogenetic analysis of SH3P sequences from selected plant lineages. SH3Ps are present in charophytes, liverworts, mosses, lycophytes, gymnosperms, and angiosperms, but not in volvocal algae, suggesting association of these proteins with phragmoplast-, but not phycoplast-based cell division. Separation of three SH3P clades, represented by SH3P1, SH3P2, and SH3P3 of A. thaliana, appears to be a seed plant synapomorphy. In the yeast two hybrid system, Arabidopsis SH3P3, but not SH3P2, binds the FH1 and FH2 domains of the formin FH5 (At5g54650), known to participate in cytokinesis, while an opposite binding specificity was found for the dynamin homolog DRP1A (At5g42080), confirming earlier findings. This suggests that the cytokinetic role of SH3P2 is not due to its interaction with FH5. Possible determinants of interaction specificity of SH3P2 and SH3P3 were identified bioinformatically.

Evolutionary Analysis of JAZ Proteins in Plants: An Approach in Search of the Ancestral Sequence.

Jasmonates are phytohormones that regulate development, metabolism and immunity. Signal transduction is critical to activate jasmonate responses, but the evolution of some key regulators such as jasmonate-ZIM domain (JAZ) repressors is not clear. Here, we identified 1065 JAZ sequence proteins in 66 lower and higher plants and analyzed their evolution by bioinformatics methods. We found that the TIFY and Jas domains are highly conserved along the evolutionary scale. Furthermore, the canonical degron sequence LPIAR(R/K) of the Jas domain is conserved in lower and higher plants. It is noteworthy that degron sequences showed a large number of alternatives from gymnosperms to dicots. In addition, ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs are displayed in all plant lineages from liverworts to angiosperms. However, the cryptic MYC2-interacting domain (CMID) domain appeared in angiosperms for the first time. The phylogenetic analysis performed using the Maximum Likelihood method indicated that JAZ ortholog proteins are grouped according to their similarity and plant lineage. Moreover, ancestral JAZ sequences were constructed by PhyloBot software and showed specific changes in the TIFY and Jas domains during evolution from liverworts to dicots. Finally, we propose a model for the evolution of the ancestral sequences of the main eight JAZ protein subgroups. These findings contribute to the understanding of the JAZ family origin and expansion in land plants.

Comprehensive Analysis and Functional Studies of WRKY Transcription Factors in Nelumbo nucifera.

The WRKY family is one of the largest transcription factor (TF) families in plants and plays central roles in modulating plant stress responses and developmental processes, as well as secondary metabolic regulations. Lotus (Nelumbo nucifera) is an aquatic crop that has significant food, ornamental and pharmacological values. Here, we performed an overview analysis of WRKY TF family members in lotus, and studied their functions in environmental adaptation and regulation of lotus benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 65 WRKY genes were identified in the lotus genome and they were well clustered in a similar pattern with their Arabidopsis homologs in seven groups (designated I, IIa-IIe, and III), although no lotus WRKY was clustered in the group IIIa. Most lotus WRKYs were functionally paired, which was attributed to the recently occurred whole genome duplication in lotus. In addition, lotus WRKYs were regulated dramatically by salicilic acid (SA), jasmonic acid (JA), and submergence treatments, and two lotus WRKYs, NnWRKY40a and NnWRKY40b, were significantly induced by JA and promoted lotus BIA biosynthesis through activating BIA biosynthetic genes. The investigation of WRKY TFs for this basal eudicot reveals new insights into the evolution of the WRKY family, and provides fundamental information for their functional studies and lotus breeding.

Mitochondrial Carriers for Aspartate, Glutamate and Other Amino Acids: A Review.

Members of the mitochondrial carrier (MC) protein family transport various molecules across the mitochondrial inner membrane to interlink steps of metabolic pathways and biochemical processes that take place in different compartments; i.e., are localized partly inside and outside the mitochondrial matrix. MC substrates consist of metabolites, inorganic anions (such as phosphate and sulfate), nucleotides, cofactors and amino acids. These compounds have been identified by in vitro transport assays based on the uptake of radioactively labeled substrates into liposomes reconstituted with recombinant purified MCs. By using this approach, 18 human, plant and yeast MCs for amino acids have been characterized and shown to transport aspartate, glutamate, ornithine, arginine, lysine, histidine, citrulline and glycine with varying substrate specificities, kinetics, influences of the pH gradient, and capacities for the antiport and uniport mode of transport. Aside from providing amino acids for mitochondrial translation, the transport reactions catalyzed by these MCs are crucial in energy, nitrogen, nucleotide and amino acid metabolism. In this review we dissect the transport properties, phylogeny, regulation and expression levels in different tissues of MCs for amino acids, and summarize the main structural aspects known until now about MCs. The effects of their disease-causing mutations and manipulation of their expression levels in cells are also considered as clues for understanding their physiological functions.

Identification of Potential Auxin-Responsive Small Signaling Peptides through a Peptidomics Approach in Arabidopsis thaliana.

Small signaling peptides (SSPs) are a class of short peptides playing critical roles in plant growth and development. SSPs are also involved in the phytohormone signaling pathway. However, identification of mature SSPs is still a technical challenge because of their extremely low concentrations in plant tissue and complicated interference by many other metabolites. Here, we report an optimized protocol to extract SSPs based on protoplast extraction and to analyze SSPs based on tandem mass spectrometry peptidomics. Using plant protoplasts as the material, soluble peptides were directly extracted into phosphate buffer. The interference of non-signaling peptides was significantly decreased. Moreover, we applied the protocol to identify potential SSPs in auxin treated wild type and auxin biosynthesis defective mutant yuc2yuc6. Over 100 potential SSPs showed a response to auxin in Arabidopsis thaliana.

Specificity of MYB interactions relies on motifs in ordered and disordered contexts.

Physical interactions between members of the MYB and bHLH transcription factor (TF) families regulate many important biological processes in plants. Not all reported MYB-bHLH interactions can be explained by the known binding sites in the R3 repeat of the MYB DNA-binding domain. Noteworthy, most of the sequence diversity of MYB TFs lies in their non-MYB regions, which contain orphan small subgroup-defining motifs not yet linked to molecular functions. Here, we identified the motif mediating interaction between MYB TFs from subgroup 12 and their bHLH partners. Unlike other known MYB-bHLH interactions, the motif locates to the centre of the predicted disordered non-MYB region. We characterised the core motif, which enabled accurate prediction of previously unknown bHLH-interacting MYB TFs in Arabidopsis thaliana, and we confirmed its functional importance in planta. Our results indicate a correlation between the MYB-bHLH interaction affinity and the phenotypic output controlled by the TF complex. The identification of an interaction motif outside R3 indicates that MYB-bHLH interactions must have arisen multiple times, independently and suggests many more motifs of functional relevance to be harvested from subgroup-specific studies.

Protein Complex Identification and quantitative complexome by CN-PAGE.

The majority of cellular processes are carried out by protein complexes. Various size fractionation methods have previously been combined with mass spectrometry to identify protein complexes. However, most of these approaches lack the quantitative information which is required to understand how changes of protein complex abundance and composition affect metabolic fluxes. In this paper we present a proof of concept approach to quantitatively study the complexome in the model plant Arabidopsis thaliana at the end of the day (ED) and the end of the night (EN). We show that size-fractionation of native protein complexes by Clear-Native-PAGE (CN-PAGE), coupled with mass spectrometry can be used to establish abundance profiles along the molecular weight gradient. Furthermore, by deconvoluting complex protein abundance profiles, we were able to drastically improve the clustering of protein profiles. To identify putative interaction partners, and ultimately protein complexes, our approach calculates the Euclidian distance between protein profile pairs. Acceptable threshold values are based on a cut-off that is optimized by a receiver-operator characteristic (ROC) curve analysis. Our approach shows low technical variation and can easily be adapted to study in the complexome in any biological system.

DELAY OF GERMINATION 1-LIKE 4 acts as an inducer of seed reserve accumulation.

More than 70% of global food supply depends on seeds. The major seed reserves, such as proteins, lipids, and polysaccharides, are produced during seed maturation. Here, we report that DELAY OF GERMINATION 1-LIKE 4 (DOGL4) is a major inducer of reserve accumulation during seed maturation. The DOGL family proteins are plant-specific proteins of largely unknown biochemical function. DOGL4 shares only limited homology in amino acid sequence with DOG1, a major regulator of seed dormancy. DOGL4 was identified as one of the outstanding abscisic acid (ABA)-induced genes in our RNA sequencing analysis, whereas DOG1 was not induced by ABA. Induction of DOGL4 caused the expression of 70 seed maturation-specific genes, even in germinating seeds, including the major seed reserves ALBUMIN, CRUCIFERIN and OLEOSIN. Although DOG1 affects the expression of many seed maturation genes, the major seed reserve genes induced by DOGL4 are not altered by the dog1 mutation. Furthermore, the reduced dormancy and longevity phenotypes observed in the dog1 seeds were not observed in the dogl4 mutants, suggesting that these two genes have limited functional overlap. Taken together, these results suggest that DOGL4 is a central factor mediating reserve accumulation in seeds, and that the two DOG1 family proteins have diverged over the course of evolution into independent regulators of seed maturation, but retain some overlapping function.

Evolutionary and Comparative Expression Analyses of TCP Transcription Factor Gene Family in Land Plants.

The plant-specific Teosinte-branched 1/Cycloidea/Proliferating (TCP) transcription factor genes are involved in plants' development, hormonal pathways, and stress response but their evolutionary history is uncertain. The genome-wide analysis performed here for 47 plant species revealed 535 TCP candidates in terrestrial plants and none in aquatic plants, and that TCP family genes originated early in the history of land plants. Phylogenetic analysis divided the candidate genes into Classes I and II, and Class II was further divided into CYCLOIDEA (CYC) and CINCINNATA (CIN) clades; CYC is more recent and originated from CIN in angiosperms. Protein architecture, intron pattern, and sequence characteristics were conserved in each class or clade supporting this classification. The two classes significantly expanded through whole-genome duplication during evolution. Expression analysis revealed the conserved expression of TCP genes from lower to higher plants. The expression patterns of Class I and CIN genes in different stages of the same tissue revealed their function in plant development and their opposite effects in the same biological process. Interaction network analysis showed that TCP proteins tend to form protein complexes, and their interaction networks were conserved during evolution. These results contribute to further functional studies on TCP family genes.

Genome-Wide Identification and Gene Expression Analysis of ABA Receptor Family Genes in Brassica juncea var. tumida.

Abscisic acid (ABA) plays important roles in multiple physiological processes, such as plant response to stresses and plant development. The ABA receptors pyrabactin resistance (PYR)/ PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) play a crucial role in ABA perception and signaling. However, little is known about the details regarding PYL family genes in Brassica juncea var. tumida. Here, 25 PYL family genes were identified in B. juncea var. tumida genome, including BjuPYL3, BjuPYL4s, BjuPYL5s, BjuPYL6s, BjuPYL7s, BjuPYL8s, BjuPYL10s, BjuPYL11s, and BjuPYL13. The results of phylogenic analysis and gene structure showed that the PYL family genes performed similar gene characteristics. By analyzing cis-elements in the promoters of those BjuPYLs, several hormone and stress related cis-elements were found. The results of gene expression analysis showed that the ABA receptor homologous genes were induced by abiotic and biotic stress. The tissue-specific gene expression patterns of BjuPYLs also suggested those genes might regulate the stem swelling during plant growth. These findings indicate that BjuPYLs are involved in plant response to stresses and organ development. This study provides valuable information for further functional investigations of PYL family genes in B. juncea var. tumida.

Relationship of GUN1 to FUG1 in chloroplast protein homeostasis.

GUN1 integrates retrograde signals in chloroplasts but the underlying mechanism is elusive. FUG1, a chloroplast translation initiation factor, and GUN1 are co-expressed at the transcriptional level, and FUG1 co-immunoprecipitates with GUN1. We used mutants of GUN1 (gun1-103) and FUG1 (fug1-3) to analyse their functional relationship at the physiological and system-wide level, the latter including transcriptome and proteome analyses. Absence of GUN1 aggravates the effects of decreased FUG1 levels on chloroplast protein translation, resulting in transiently more pronounced phenotypes regarding photosynthesis, leaf colouration, growth and cold acclimation. The gun1-103 mutation also enhances variegation in the var2 mutant, increasing the fraction of white sectors, while fug1-3 suppresses the var2 phenotype. The transcriptomes of fug1-3 and gun1-103 plants are very similar, but absence of GUN1 alone has almost no effect on protein levels, whereas steady-state levels of chloroplast proteins are markedly decreased in fug1-3. In fug1 gun1 double mutants, effects on transcriptomes and particularly on proteomes are enhanced. Our results show that GUN1 function becomes critical when chloroplast proteostasis is perturbed by decreased rates of synthesis (fug1) or degradation (var2) of chloroplast proteins, or by low temperatures. The functions of FUG1 and GUN1 appear to be related, corroborating the view that GUN1 helps to maintain chloroplast protein homeostasis (proteostasis).

AtBUD13 affects pre-mRNA splicing and is essential for embryo development in Arabidopsis.

Pre-mRNA splicing is an important step for gene expression regulation. Yeast Bud13p (bud-site selection protein 13) regulates the budding pattern and pre-mRNA splicing in yeast cells; however, no Bud13p homologs have been identified in plants. Here, we isolated two mutants that carry T-DNA insertions at the At1g31870 locus and shows early embryo lethality and seed abortion. At1g31870 encodes an Arabidopsis homolog of yeast Bud13p, AtBUD13. Although AtBUD13 homologs are widely distributed in eukaryotic organisms, phylogenetic analysis revealed that their protein domain organization is more complex in multicellular species. AtBUD13 is expressed throughout plant development including embryogenesis and AtBUD13 proteins is localized in the nucleus in Arabidopsis. RNA-seq analysis revealed that AtBUD13 mutation predominantly results in the intron retention, especially for shorter introns (≤100 bases). Within this group of genes, we identified 52 genes involved in embryogenesis, out of which 22 are involved in nucleic acid metabolism. Our results demonstrate that AtBUD13 plays critical roles in early embryo development by effecting pre-mRNA splicing.

Genome-wide analysis of the Chinese cabbage IQD gene family and the response of BrIQD5 in drought resistance.

KEY MESSAGE: Thirty-five IQD genes were identified and analysed in Chinese cabbage and BrIQD5 transgenic plants enhanced the drought resistance of plants. The IQD (IQ67-domain) family plays an important role in various abiotic stress responses in plant species. However, the roles of IQD genes in the Chinese cabbage response to abiotic stress remain unclear. Here, 35 IQD genes, from BrIQD1 to BrIQD35, were identified in Chinese cabbage (Brassica rapa ssp. pekinensis). Based on the phylogenetic analysis, these genes were clustered into three subfamilies (I-III), and members within the same subfamilies shared conserved exon-intron distribution and motif composition. The 35 BrIQD genes were unevenly distributed on 9 of the 10 chromosomes with 4 segmental duplication events. Ka/Ks ratios showed that the duplicated BrIQDs had mainly experienced strong purifying selection. Quantitative real-time polymerase chain reaction of 35 BrIQDs under PEG6000 indicated that BrIQD5 was significantly induced by PEG6000. To verify BrIQD5 function, BrIQD5 was heterologously overexpressed in tobacco and was silenced in Chinese cabbage. BrIQD5-overexpressed plants showed more tolerance to drought stress than wild-type plants, while BrIQD5-silenced plants in Chinese cabbage showed decreased drought tolerance. Additionally, six BrIQD5 potential interactive proteins were isolated by the yeast two-hybrid assay, including BrCaMa, BrCaMb and four other stress-related proteins. Motif IQ1 of BrIQD5 is important for the interaction with BrCaMa and BrCaMb, and the isoleucine in motif IQ1 is an essential amino acid for calmodulin binding to BrIQD5. The identification and cloning of the new Chinese cabbage drought tolerance genes will promote the drought-resistant breeding of Chinese cabbage and help to better understand the mechanism of IQD involved in the drought tolerance of plants.