Pineapple SWEET10 is a glucose transporter.

SWEET transporters are a unique class of sugar transporters that play vital roles in various developmental and physiological processes in plants. While the functions of SWEETs have been well established in model plants such as Arabidopsis, their functions in economically important fruit crops like pineapple have not been well studied. Here we aimed to investigate the substrate specificity of pineapple SWEETs by comparing the protein sequences of known glucose and sucrose transporters in Arabidopsis with those in pineapple. Our genome-wide approach and 3D structure comparison showed that the Arabidopsis SWEET8 homolog in pineapple, AcSWEET10, shares similar sequences and protein properties responsible for glucose transport. To determine the functional conservation of AcSWEET10, we tested its ability to complement glucose transport mutants in yeast and analyzed its expression in stamens and impact on the microspore phenotype and seed set in transgenic Arabidopsis. The results showed that AcSWEET10 is functionally equivalent to AtSWEET8 and plays a critical role in regulating microspore formation through the regulation of the Callose synthase5 (CalS5), which highlights the importance of SWEET transporters in pineapple. This information could have important implications for improving fruit crop yield and quality by manipulating SWEET transporter activity.

The genome size, chromosome number and the seed adaption to long-distance dispersal of Ipomoea pes-caprae (L.).

Ipomoeapes-caprae (L.) (IPC) is a common species in tropical and subtropical coastal areas and one of the world's most widely distributed plants. It has attracted researchers for its outstanding biological, ecological and medicinal values. It has been reported that the genetic diversity of IPCs located on different continents is very low because of their frequent gene flow. During the long journey of evolution, every aspect of the plant morphologies has evolved to the best adaptivity to the environment, seeking their survival and progeny expansion. However, the fundamental genetic characteristics of IPC and how their seed adapted to the success of population expansion remain unknown. In this study, the fundamental genetic characteristics, including the genome size and the chromosome number of IPC, were investigated. The results showed that IPC's genome size is approximately 0.98-1.08 GB, and the chromosome number is 2n=30, providing the basic information for further genome analysis. In order to decipher the long-distance dispersal secret of this species, the fruit and seed developments, seed morphology, and seed germination were extensively investigated and described. The results showed an exquisite adaptive mechanism of IPC seeds to fulfil the population expansion via ocean currents. The large cavity inside the seeds and the dense tomenta on the surface provide the buoyancy force for the seeds to float on the seawater. The hard seed coats significantly obstructed the water absorption, thus preventing the seed from germination during the dispersal. Meanwhile, the fully developed embryos of IPC also have physiological dormancy. The physical and physiological characteristics of IPC seeds provide insight into the mechanism of their long-distance dispersal across the oceans. Moreover, based on morphological observation and semi-section microscopy, the development pattern of IPC glander trichomes was described, and their physiological functions were also discussed.

Identification and expression analysis of pineapple sugar transporters reveal their role in the development and environmental response.

In plants, sugars are required for several essential functions, including growth, storage, signaling, defense and reproduction. Sugar transporters carry out the controlled movement of sugars from source (leaves) to sink (fruits and roots) tissues and determine the overall development of the plant. Various types of sugar transporter families have been described in plants, including sucrose transporters (SUC/SUT), monosaccharide transporter (MST) and SWEET (from "Sugar Will Eventually be Exported Transporters"). However, the information about pineapple sugar transporters is minimal. This study systematically identified and classified 45 MST and 4 SUC/SUT genes in the pineapple genome. We found that the expression patterns of sugar transporter genes have a spatiotemporal expression in reproductive and vegetative tissues indicating their pivotal role in reproductive growth and development. Besides, different families of sugar transporters have a diel expression pattern in photosynthetic and non-photosynthetic tissues displaying circadian rhythm associated participation of sugar transporters in the CAM pathway. Moreover, regulation of the stress-related sugar transporters during cold stress indicates their contribution to cold tolerance in pineapple. Heterologous expression (yeast complementation assays) of sugar transporters in a mutant yeast strain suggested that SUT1/2 have the ability to transport sucrose, and STP13, STP26, pGlcT-L2 and TMT4 are able to transport glucose, whereas SWEET11/13 transport both sucrose and fructose. The information provided here would help researchers further explore the underlying molecular mechanism involved in the sugar metabolism of pineapple.

AcCIPK5, a pineapple CBL-interacting protein kinase, confers salt, osmotic and cold stress tolerance in transgenic Arabidopsis.

Plant-specific calcineurin B-like proteins (CBLs) and their interacting kinases, CBL-interacting protein kinases (CIPKs) module, are essential for dealing with various biotic and abiotic stress. The kinases (CIPKs) of this module have been well studied in several plants; however, the information about pineapple CIPKs remains limited. To understand how CIPKs function against environmental cues in pineapple, the CIPK5 gene of pineapple was cloned and characterized. The phylogenetic analyses revealed that AcCIPK5 is homologous to the CIPK12 of Arabidopsis and other plant species. Quantitative real-time PCR (qRT-PCR) analysis revealed that AcCIPK5 responds to multiple stresses, including osmotic, salt stress, heat and cold. Under optimal conditions, AcCIPK5 gets localized to the cytoplasm and cell membrane. The ectopic expression of AcCIPK5 in Arabidopsis improved the germination under osmotic and salt stress. Furthermore, AcCIPK5 positively regulated osmotic, drought, salt and cold tolerance and negatively regulated heat and fungal stress in Arabidopsis. Besides, the expression of AcCIPK impacted ABA-related genes and ROS homeostasis. Overall, the present study demonstrates that AcCIPK5 contributes to multiple stress tolerance and has the potential to be utilized in the development of stress-tolerant crops.

Big Role of Small RNAs in Female Gametophyte Development.

In living organisms, sexual reproduction relies on the successful development of the gametes. Flowering plants produce gametes in the specialized organs of the flower, the gametophytes. The female gametophyte (FG), a multicellular structure containing female gametes (egg cell and central cell), is often referred to as an embryo sac. Intriguingly, several protein complexes, molecular and genetic mechanisms participate and tightly regulate the female gametophyte development. Recent evidence indicates that small RNA (sRNA) mediated pathways play vital roles in female gametophyte development and specification. Here, we present an insight into our understanding and the recent updates on the molecular mechanism of different players of small RNA-directed regulatory pathways during ovule formation and growth.

Interspecific complementation-restoration of phenotype in Arabidopsis cuc2cuc3 mutant by sugarcane CUC2 gene.

BACKGROUND: In plants, a critical balance between differentiation and proliferation of stem cells at the shoot apical meristem zone is essential for proper growth. The spatiotemporal regulation of some crucial genes dictates the formation of a boundary within and around budding organs. The boundary plays a pivotal role in distinguishing one tissue type from another and provides a defined shape to the organs at their developed stage. NAM/CUC subfamily of the NAC transcription factors control the boundary formation during meristematic development. RESULTS: Here, we have identified the CUP-SHAPED COTYLEDON (CUC) genes in sugarcane and named SsCUC2 (for the orthologous gene of CUC1 and CUC2) and SsCUC3. The phylogenetic reconstruction showed that SsCUCs occupy the CUC2 and CUC3 clade together with monocots, whereas eudicot CUC2 and CUC3 settled separately in the different clade. The structural analysis of CUC genes showed that most of the CUC3 genes were accompanied by an intron gain during eudicot divergence. Besides, the study of SsCUCs expression in the RNA-seq obtained during different stages of ovule development revealed that SsCUCs express in developing young tissues, and the expression of SsCUC2 is regulated by miR164. We also demonstrate that SsCUC2 (a monocot) could complement the cuc2cuc3 mutant phenotype of Arabidopsis (eudicot). CONCLUSIONS: This study further supports that CUC2 has diverged in CUC1 and CUC2 during the evolution of monocots and eudicots from ancestral plants. The functional analysis of CUC expression patterns during sugarcane ovule development and ectopic expression of SsCUC2 in Arabidopsis showed that SsCUC2 has a conserved role in boundary formation. Overall, these findings improve our understanding of the functions of sugarcane CUC genes. Our results reveal the crucial functional role of CUC genes in sugarcane.

TRM61 is essential for Arabidopsis embryo and endosperm development.

Post-transcriptional modifications of tRNA molecules play crucial roles in gene expression and protein biosynthesis. Across the genera, methylation of tRNAs at N1 of adenosine 58 (A58) by AtTRM61/AtTRM6 complex plays a critical role in maintaining the stability of initiator methionyl-tRNA (tRNAiMet). Recently, it was shown that mutation in AtTRM61 or AtTRM6 leads to seed abortion. However, a detailed study about the AtTRM61/AtTRM6 function in plants remains vague. Here, we found that AtTRM61 has a conserved functional structure and possesses conserved binding motifs for cofactor S-adenosyl-L-methionine (AdoMet). Mutations of the complex subunits AtTRM61/AtTRM6 result in embryo and endosperm developmental defects. The endosperm and embryo developmental defects were conditionally complemented by Attrm61-1/ + FIS2pro::AtTRM61 and Attrm61-1/ + ABI3pro::AtTRM61 indicating that AtTRM61 is required for early embryo and endosperm development. Besides, the rescue of the fertility defects in trm61/ + by overexpression of initiator tRNA suggests that AtTRM61 mutation could diminish tRNAiMet stability. Moreover, using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays, we showed that AtMPK4 physically interacts with AtTRM61. The data presented here suggest that AtTRM61 has a conserved structure and function in Arabidopsis. Also, AtTRM61 may be required for tRNAiMet stability, embryo and endosperm development.

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.

BACKGROUND: Transcription factors (TFs) are essential regulators of growth and development in eukaryotes. Basic-helix-loop-helix (bHLHs) is one of the most significant TFs families involved in several critical regulatory functions. Cryptochrome-interacting bHLH (CIB) and cryptochromes form an extensive regulatory network to mediate a plethora of pathways. Although bHLHs regulate critical biological processes in plants, the information about pineapple bHLHs remains unexplored. RESULTS: Here, we identified a total of 121 bHLH proteins in the pineapple genome. The identified genes were renamed based on the ascending order of their gene ID and classified into 18 subgroups by phylogenetic analysis. We found that bHLH genes are expressed in different organs and stages of pineapple development. Furthermore, by the ectopic expression of AcCIB2 in Arabidopsis and complementation of Atcib2 mutant, we verified the involvement of AcCIB2 in photomorphogenesis and abiotic stress response. CONCLUSIONS: Our findings revealed that AcCIB2 plays an essential role in flowering time regulation and abiotic stress response. 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.

SWR1 Chromatin Remodeling Complex: A Key Transcriptional Regulator in Plants.

The nucleosome is the structural and fundamental unit of eukaryotic chromatin. The chromatin remodeling complexes change nucleosome composition, packaging and positioning to regulate DNA accessibility for cellular machinery. SWI2/SNF2-Related 1 Chromatin Remodeling Complex (SWR1-C) belongs to the INO80 chromatin remodeling family and mainly catalyzes the exchange of H2A-H2B with the H2A.Z-H2B dimer. The replacement of H2A.Z into nucleosomes affects nucleosome stability and chromatin structure. Incorporation of H2A.Z into the chromatin and its physiochemical properties play a key role in transcriptional regulation during developmental and environmental responses. In Arabidopsis, various studies have uncovered several pivotal roles of SWR1-C. Recently, notable progress has been achieved in understanding the role of SWR1-C in plant developmental and physiological processes such as DNA damage repair, stress tolerance, and flowering time. The present article introduces the SWR1-C and comprehensively reviews recent discoveries made in understanding the function of the SWR1 complex in plants.

Identification of SWI2/SNF2-Related 1 Chromatin Remodeling Complex (SWR1-C) Subunits in Pineapple and the Role of Pineapple SWR1 COMPLEX 6 (AcSWC6) in Biotic and Abiotic Stress Response.

Chromatin remodeling complex orchestrates numerous aspects of growth and development in eukaryotes. SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is a member of the SWI/SNF ATPase-containing chromatin remodeling complex responsible for the exchange of H2A for H2A.Z. In plants, SWR1-C plays a crucial role by transcriptionally regulating numerous biological and developmental processes. However, SWR1-C activity remains obscure in pineapple. Here, we aim to identify the SWR1-C subunits in pineapple. By genome-wide identification, we found a total of 11 SWR1-C subunits in the pineapple. The identified SWR1-C subunits were named and classified based on the sequence similarity and phylogenetic analysis. RNA-Seq analysis showed that pineapple SWR1-C subunits are expressed differentially in different organs and at different stages. Additionally, the qRT-PCR of pineapple SWR1-C subunits during abiotic stress exposure showed significant changes in their expression. We further investigated the functions of pineapple SWR1 COMPLEX 6 (AcSWC6) by ectopically expressing it in Arabidopsis. Interestingly, transgenic plants ectopically expressing AcSWC6 showed susceptibility to fungal infection and enhanced resistance to salt and osmotic stress, revealing its involvement in biotic and abiotic stress. Moreover, the complementation of mutant Arabidopsisswc6 by pineapple SWC6 suggested the conserved function of SWC6 in plants.

Genome-Wide Identification and Expression Profiling of CBL-CIPK Gene Family in Pineapple (Ananas comosus) and the Role of AcCBL1 in Abiotic and Biotic Stress Response.

Ca2+ serves as a ubiquitous second messenger regulating several aspects of plant growth and development. A group of unique calcium sensor proteins, calcineurin B-like (CBL), interact with CBL-interacting protein kinases (CIPKs) to decode the Ca2+ signature inside the cell. Although CBL-CIPK signaling toolkit has been shown to play significant roles in the responses to numerous stresses in different plants, the information about pineapple CBL-CIPK remains obscure. In the present study, a total of eight AcCBL and 21 AcCIPK genes were identified genome-wide in pineapple. The identified genes were renamed on the basis of gene ID in ascending order and phylogenetic analysis divided into five groups. Transcriptomic data analysis showed that AcCBL and AcCIPK genes were expressed differentially in different tissues. Further, the expression analysis of AcCBL1 in different tissues showed significant changes under various abiotic stimuli. Additionally, the ectopic expression of AcCBL1 in Arabidopsis resulted in enhanced tolerance to salinity, osmotic, and fungal stress. The present study revealed the crucial contribution of the CBL-CIPK gene in various biological and physiological processes in pineapple.

Ectopic Expression of Cold Responsive LlaCIPK Gene Enhances Cold Stress Tolerance in Nicotiana tabacum.

Low-temperature stress severely affects the growth, development, and geographical distribution of various crop plants, resulting in significant economic loss to producers. In a quest to identify cold-regulated genes, we constructed a cDNA suppression subtractive library from a high altitude adapted ecotype of Lepidium. We cloned a cold-induced gene LlaCIPK from the subtracted cDNA library which gave homology to Arabidopsis CIPK15 gene. The predicted 3D structure of LlaCIPK protein also showed homology with Arabidopsis CIPK protein. Quantitative real-time PCR analysis in Lepidium seedlings exposed to 6 h of cold stress shows a 3-fold increase in the expression of LlaCIPK transcript. The expression of LlaCIPK was also differentially regulated by ethylene, CaCl2, ABA, and SA treatments. Ethylene and CaCl2 treatments up regulated LlaCIPK expression, whereas ABA and SA treatments down regulated the LlaCIPK expression. Transgenic plants overexpressing LlaCIPK gene under constitutive promoter show an increased level of proline and cell membrane stability. Taken together, our results suggest that the LlaCIPK contributes to the cold-response pathway in Lepidium plants.

Isolation and characterization of cold responsive NAC gene from Lepidium latifolium.

Cold stress is one of the major limiting factor in crop productivity. Plants growing in colder regions acclimatize to severe conditions owing to the presence of 'cold stress tolerant genes'. Isolation and functional characterization of these genes are important before their exploitation in modern agricultural practices. Here, we have cloned full length NAC gene (1,388 bp) from Lepidium latifolium (LlaNAC). This gene belongs to NAP sub-group which also includes ANAC056 of Arabidopsis thaliana, nearest relative of LlaNAC. Upstream analysis and microarray data analysis of ANAC056 suggested that LlaNAC might also be ABA-regulated. However, quantitative transcript expression analysis revealed that LlaNAC transcript upregulated by cold stress and downregulated in response to varying concentrations of abscisic acid, salicylic acid, calcium chloride and ethylene. There is also a possibility that the gene may be getting regulated by a pathway whose components are still unknown. Any further investigations to understand the mechanism of regulation of LlaNAC gene expression are likely to find immense importance in plant biotechnology and crop improvement.