Genome-wide study of pineapple (Ananas comosus L.) bHLH transcription factors indicates that cryptochrome-interacting bHLH2 (AcCIB2) participates in flowering time regulation and abiotic stress response

- Basic- helix-loop-helix (bHLHs) is one of the most significant TFs families involved in several critical regulatory functions..
- Results: Here, we identified a total of 121 bHLH proteins in the pineapple genome.
- We found that bHLH genes are expressed in different organs and stages of pineapple development.
- The present study provides additional insights into the current knowledge of bHLH genes and suggests their potential role in various biological processes during pineapple development..
- TFs could repress or activate the expression of their target genes, resulting in the regulation of the development and physiological response.
- TFs could repress or activate the tar- get gene expression resulting in the regulation of develop- ment and response [1, 2].
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- The basic region is found at the N-terminus of the domain and modulates DNA binding, whereas the HLH region of the domain facilitates dimerization through protein- protein interaction [6].
- CRYs are photolyase-associated blue-light receptors, and they interact with different proteins in the presence of blue- light to mediate a plethora of functions, such as inhibition of hypocotyl elongation and flowering initiation .
- CIBs act redundantly in the CRY-CIB pathway to promote flower induction [9].
- Identification and characterization of pineapple bHLH genes.
- The bHLH genes of pineapple showed high similarity to those in Arabidopsis.
- We further characterized the pineapple bHLH proteins based on their molecular weight, isoelectric point, amino acid, and open reading frame (ORF) length, respectively (Additional Table S1)..
- AcbHLH15 (Aco001136) has the lowest molecular weight of 1.04 kDa among the pineapple bHLH proteins.
- The pineapple bHLH proteins also have different isoelectric point values, ranging from AcbHLH30 (Aco002151) with the highest of 10.76, and AcbHLH53 recording the smallest value of 4.73.
- The exon-intron analysis suggests that most of the AcbHLH possess introns, and with forty-nine introns, AcbHLH17 had the maximum number of introns.
- Interestingly, all the AcCIBs were in the group XII with Arabidopsis CIBs.
- The pineapple CIB genes grouped with their corresponding Arabidopsis homolog suggest that they may have a similar biological function in photomorpho- genesis and developmental responses..
- We then studied the distribution of AcbHLHs on pine- apple chromosomes and found that pineapple bHLH genes are distributed unevenly on 23 linkage groups (LG).
- Only two pineapple linkage groups, LG 22 and LG 24, do not possess bHLH genes.
- Few linkage groups have a higher density of bHLH genes (up to 11 genes), whereas few have only one, and all the AcbHLHs are dis- tributed on different LGs (Fig.
- The pineapple bHLH domain analysis indicates that the average length of the AcbHLH domain was approxi- mately 50 aa, which ranged from 34 to 56 aa (Fig.
- Further, the study of conserved motif distribution of AcbHLH superfamily using the MEME program resulted in the identification of ten dif- ferent motifs distributed among AcbHLH proteins.
- 1 Phylogenetic tree showing the relationship between bHLH genes of pineapple and Arabidopsis.
- Red circles represent the pineapple CIB genes and the green represents Arabidopsis CIB genes.
- 2 Chromosomal locations of pineapple bHLH genes.
- The bHLH genes of pineapple were mapped to different chromosomes using MapChart.
- Expression profile of pineapple bHLH genes in pineapple We performed the expression profile analysis of pine- apple bHLH genes using RNA-seq from various develop- mental stages of different organs to understand their possible functions in pineapple.
- The altered expression patterns of the majority of AcbHLH in the selected sam- ples suggest that the pineapple bHLH genes play different roles in a specific organ or developmental stage.
- Low expressed bHLH genes were gathered together in group I, highly expressed genes formed group II and the moderately expressed genes formed group III (Fig.
- Depending on the developmental requirements, the expression of the AcbHLHs was a stage and/or organ- specific.
- For example, AcbHLH113, a homolog of ABORTED MICROSPORES (AMS), had relatively high expression levels in the different stages of stamen develop- ment, indicating that it might be playing a crucial role in pineapple anther development (Fig.
- Most of the pine- apple CIB genes except AcCIB1 (AcbHLH71) were in group II, displaying a high expression level in the stages of flower and fruit development, suggesting that the pine- apple AcCIB1 may not be the primary regulator of flower- ing.
- CIB2 (AcbHLH8), CIB3 (AcbHLH107), CIB4 (AcbHLH92) and CIB5 (AcbHLH12) are in group II with highly expressed bHLH genes.
- The observation of 7-day old roots of transgenic plants under a confocal microscope showed that AcCIB2-GFP is a nuclear protein, and it localizes in the nu- cleus (Fig.
- While the plants ectopi- cally expressing AcCIB2 do not show any significant differ- ences in the flowering time and produced flower between 22 to 24 days, similar to wild-type plants (Fig.
- We found that the ex- pression level of SOC1 and FT was significantly higher in the cib2 mutant, whereas the expression of CO was reduced compared to Col-0 plants (Fig.
- The complemented plants of cib2 mutant by AcCIB2 did not show any signifi- cant change in the expression of selected genes compared to wild type, suggesting that AcCIB2 may have a conserved role in plants.
- However, the ectopic expression of the AcCIB2 changed the expression of CRY-CIB genes, and the expres- sion of CRY1, CIB1 and CIB5 were significantly altered, sug- gesting that the CRY-CIB genes may be working redundantly in the pathways (Fig.
- Ectopic expression of AcCIB2 enhances abiotic stress resistance.
- 3 Sequence characteristics of the bHLH domains.
- In the germination assay, we found that the Atcib2 mutant displays susceptibility to salinity and osmotic.
- 4 Expression profiles of the pineapple bHLH genes.
- The right side of the figure shows the scale.
- Details of the samples are mentioned at the bottom of each lane: sepal Se1 – Se4, gynoecium Gy1- Gy7, ovule Ov1 – Ov7, petal Pe1 – Pe3, stamen St1 – St6, fruit ‘ Fr_S1 – Fr_S7.
- Overall, these findings approve the role of cryptochromes in abiotic stress response..
- The availability of the sequenced genome of pineapple serves as a great genetic resource for studying the gene families [29].
- Here, we identified 121 bHLH genes that form a large family in pineapple.
- Similarly, pineapple also pos- sesses a large number of bHLH proteins suggesting their dis- pensable role in the pineapple evolution and development..
- Previous studies suggest that the two helices of the bHLH domain fulfill the DNA binding prerequisite by forming the homo or heterodimers between the bHLH proteins.
- Pineapple bHLH proteins also.
- share similar conserved domains and amino acid sequences with the Arabidopsis proteins in the same cluster..
- We found different exon-intron structures in AcbHLH super- family, some of the genes have no introns in their struc- ture, and some are intron rich, in contrast, some have few numbers of introns (Additional Figure S2, Additional Table S1).
- 1), the pineapple bHLH also formed a group with their Arabidopsis homologs.
- One of the essential class of photo-receptors involved in flowering initiation is cryptochromes (CRYs) and their interacting proteins.
- Most of the photoreceptors signaling mechanisms have been reported in Arabidopsis [15], and recently in tomato [17].
- AcCIB2-GFP localization in the nucleus of seven-day-old roots of transgenic Arabidopsis plants.
- The finding that AcCIB2 expresses in the majority of developmental stages and could be involved in flower development led us further to study the function of AcCIB2.
- Plants were then photographed after 20 days of the transfer.
- level of FT, SOC1 and other CIBs in the Atcib2 and the transgenic Arabidopsis plants with pineapple CIB2.
- Con- sistently, the transcript level of FT and SOC1 genes chan- ged significantly in the mutant.
- Taken together, the present study provides a platform to study the pineapple AcbHLH genes.
- Future studies with the specific AcbHLHs that are involved in the particular pathway will further clarify how AcbHLHs regulate the re- sponse to biotic and abiotic stresses in pineapple..
- In this study, a comprehensive investigation of bHLH genes was performed, and 121 AcbHLH genes were identi- fied in the pineapple genome.
- Pineapple bHLH genes were further classified into 18 subfamilies.
- Besides, the functional characterization of AcbHLH8 (AcCIB2) shows the conserved functional role of bHLH genes in photo- morphogenesis and response abiotic stress.
- Overall, this study provides important information about the potential functions of the AcbHLHs, especially the AcCIB2 role in flowering, which is an essential trait for crop breeding..
- The plated seeds were then kept for stratification in the dark at 4 °C for 48 h.
- Using the SMART tool (smart.embl-heidelberg.de), the completeness and existence of the core domain in all the sequences were then verified [43]..
- The quality of the libraries was determined on the Agilent Bioanalyzer system and sequenced on a HiSeq2500 sequencing instrument using 150 bp paired-end protocol.
- The published pineapple genome was used as the reference genome [29], and reads were aligned to the pineapple genome by using TopHat v2.1.1 [48].
- The fold change in the expression of genes was determined using the Livak method ( 2 −ΔΔC T.
- and the pineapple ef1 α gene was used as the internal control [50].
- The properties of bHLH genes in pineapple..
- Exon-intron structure of pineapple bHLH genes.
- The sequence information for each motif is provided in the bottom box.
- The expression profile of pineapple bHLH genes in different tissue and developmental stages.
- Details of the samples are: sepal Se1 – Se4, gynoecium Gy1- Gy7, ovule Ov1 – Ov7, petal.
- List of primers used in the present study..
- Protein sequences analysed in the present study..
- DNA sequences analysed in the present study..
- We thank all members of Qin lab for their assistance in the experiments..
- provided the materials and assisted in the revision of the manuscript.
- All of the authors read and approved the final manuscript..
- The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript..
- The datasets generated and/or analysed during the current study are available in the NCBI SRA repository under accession number PRJEB38680 [51]..
- Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae.
- Genome- wide identification and characterization of the potato bHLH transcription factor family.
- Multiple bHLH proteins form heterodimers to mediate CRY2-dependent regulation of flowering-time in Arabidopsis.
- Genome-wide survey of the bHLH super gene family in Brassica napus..
- Genome-wide analysis of the Cryptochrome gene family in plants..
- Arabidopsis CRY2 and ZTL mediate blue-light regulation of the transcription factor CIB1 by distinct mechanisms.
- Genome-wide analysis of the basic helix-loop-helix (bHLH) transcription factor family in maize.
- The pineapple genome and the evolution of CAM photosynthesis.
- Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis.
- Cryptochrome-related abiotic stress responses in plants.
- From the cover: a role for Arabidopsis cryptochromes and COP1 in the regulation of stomatal opening..
- 20 years of the SMART protein domain annotation resource

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