VIP1 and its close homologs confer mechanical stress tolerance in Arabidopsis leaves.

VIP1, an Arabidopsis thaliana basic leucine zipper transcription factor, and its close homologs are imported from the cytoplasm to the nucleus when cells are exposed to mechanical stress. They bind to AGCTG (G/T) and regulate mechanical stress responses in roots. However, their role in leaves is unclear. To clarify this, mutant lines (QM1 and QM2) that lack the functions of VIP1 and its close homologs (bZIP29, bZIP30 and PosF21) were generated. Brushing more severely damaged QM1 and QM2 leaves than wild-type leaves. Genes regulating stress responses and cell wall properties were downregulated in brushed QM2 leaves and upregulated in brushed VIP1-GFP-overexpressing (VIP1-GFPox) leaves compared to wild-type leaves in a transcriptome analysis. The VIP1-binding sequence AGCTG (G/T) was enriched in the promoters of genes downregulated in brushed QM2 leaves compared to wild-type leaves and in those upregulated in brushed VIP1-GFPox leaves. Calmodulin-binding transcription activators (CAMTAs) are known regulators of mechanical stress responses, and the CAMTA-binding sequence CGCGT was enriched in the promoters of genes upregulated in the brushed QM2 leaves and in those downregulated in the brushed VIP1-GFPox leaves. These findings suggest that VIP1 and its homologs upregulate genes via AGCTG (G/T) and influence CAMTA-dependent gene expression to enhance mechanical stress tolerance in leaves.

Metagenomic Analyses of Viruses in the Orchid Mycorrhizal Interaction Using Improved Assemble Tools.

In nature, mycorrhizal association with soil-borne fungi is indispensable for orchid species. Compatible mycorrhizal fungi form endo-mycorrhizal structures in orchid cells, and the fungal structures are digested in orchid cells to be supplied to orchids as nutrition. Because orchid seeds lack the reserves for germination, they keep receiving nutrition through mycorrhizal formation from seed germination until nonphotosynthetic young seedlings develop leaves and become photoautotrophic. Seeds of all orchids germinate with the help of their own fungal partners, and therefore, specific partnership has been acquired in a long evolutionary history between orchids and fungi. Assuming that horizontal transmission of viruses may occur in such a close relationship, we are focusing on viruses that infect orchids and their mycorrhizal fungi. We prepared aseptically germinated orchid plants (i.e., fungi-free plants) together with pure-cultured fungal isolates and conducted transcriptome analyses (RNA-seq) by next-generation sequencing (NGS) approach. To reconstruct virus-related sequences that would have been present in the RNA sample of interest, de novo assembly process is required using short read sequences obtained from RNA-seq. In the previous version of our protocol (see Viral Metagenomics, first edition 2018), virus searches were conducted using contig sets constructed by a single assembler, but this time we devised a method to construct more reliable contigs using multiple assemblers and again reinvestigated that viruses could be detected. Because the virus detection efficiency and number of detected virus species clearly differed depending on the assembly pipeline and the number of the input data, multiple methods should be used to identify viral infection, if possible.

Transcriptomic Analyses Reveal the Role of Cytokinin and the Nodal Stem in Microtuber Sprouting in Potato (Solanum tuberosum L.).

In potatoes, tuber secondary growth, especially sprouting, deforms the tubers and severely lowers their commercial value. Tuber sprouting is induced by signal substances, such as gibberellin (GA), which are transported to the tuber from the plant body. The molecular mechanism underlying GA-induced sprouting remains ambiguous. Here, we tried to recreate tuber secondary growth using in vitro stemmed microtubers (MTs) (with the nodal stem attached) and MT halves (with the nodal stem entirely removed). Our experiments showed that GA alone could initiate the sprouting of stemmed microtubers; however, GA failed to initiate MT halves unless 6-benzyladenine, a synthetic cytokinin CK, was co-applied. Here, we analyzed the transcriptional profiles of sprouting buds using these in vitro MTs. RNA-seq analysis revealed a downregulation of cytokinin-activated signaling but an upregulation of the "Zeatin biosynthesis" pathway, as shown by increased expression of CYP735A, CISZOG, and UGT85A1 in sprouting buds; additionally, the upregulation of genes, such as IAA15, IAA22, and SAUR50, associated with auxin-activated signaling and one abscisic acid (ABA) negative regulator, PLY4, plays a vital role during sprouting growth. Our findings indicate that the role of the nodal stem is synonymous with CK in sprouting growth, suggesting that CK signaling and homeostasis are critical to supporting GA-induced sprouting. To effectively control tuber sprouting, more effort is required to be devoted to these critical genes.

Construction of a de novo assembly pipeline using multiple transcriptome data sets from Cypripedium macranthos (Orchidaceae).

The family Orchidaceae comprises the most species of any monocotyledonous family and has interesting characteristics such as seed germination induced by mycorrhizal fungi and flower morphology that co-adapted with pollinators. In orchid species, genomes have been decoded for only a few horticultural species, and there is little genetic information available. Generally, for species lacking sequenced genomes, gene sequences are predicted by de novo assembly of transcriptome data. Here, we devised a de novo assembly pipeline for transcriptome data from the wild orchid Cypripedium (lady slipper orchid) in Japan by mixing multiple data sets and integrating assemblies to create a more complete and less redundant contig set. Among the assemblies generated by combining various assemblers, Trinity and IDBA-Tran yielded good assembly with higher mapping rates and percentages of BLAST hit contigs and complete BUSCO. Using this contig set as a reference, we analyzed differential gene expression between protocorms grown aseptically or with mycorrhizal fungi to detect gene expressions required for mycorrhizal interaction. A pipeline proposed in this study can construct a highly reliable contig set with little redundancy even when multiple transcriptome data are mixed, and can provide a reference that is adaptable to DEG analysis and other downstream analysis in RNA-seq.

Functional characterization and vacuolar localization of fructan exohydrolase derived from onion (Allium cepa).

Fructans such as inulin and levan accumulate in certain taxonomic groups of plants and are a reserve carbohydrate alternative to starch. Onion (Allium cepa L.) is a typical plant species that accumulates fructans, and it synthesizes inulin-type and inulin neoseries-type fructans in the bulb. Although genes for fructan biosynthesis in onion have been identified so far, no genes for fructan degradation had been found. In this study, phylogenetic analysis predicted that we isolated a putative vacuolar invertase gene (AcpVI1), but our functional analyses demonstrated that it encoded a fructan 1-exohydrolase (1-FEH) instead. Assessments of recombinant proteins and purified native protein showed that the protein had 1-FEH activity, hydrolyzing the ß-(2,1)-fructosyl linkage in inulin-type fructans. Interestingly, AcpVI1 had an amino acid sequence close to those of vacuolar invertases and fructosyltransferases, unlike all other FEHs previously found in plants. We showed that AcpVI1 was localized in the vacuole, as are onion fructosyltransferases Ac1-SST and Ac6G-FFT. These results indicate that fructan-synthesizing and -degrading enzymes are both localized in the vacuole. In contrast to previously reported FEHs, our data suggest that onion 1-FEH evolved from a vacuolar invertase and not from a cell wall invertase. This demonstrates that classic phylogenetic analysis on its own is insufficient to discriminate between invertases and FEHs, highlighting the importance of functional markers in the nearby active site residues.

Revision of the relationship between anther morphology and pollen sterility by cold stress at the booting stage in rice.

BACKGROUND AND AIMS: Cold stress in rice (Oryza sativa) plants at the reproductive stage prevents normal anther development and causes pollen sterility. Tapetum hypertrophy in anthers has been associated with pollen sterility in response to cold at the booting stage. Here, we re-examined whether the relationships between anther abnormality and pollen sterility caused by cold stress at the booting stage in rice can be explained by a monovalent factor such as tapetum hypertrophy. METHODS: After exposing plants to a 4-d cold treatment at the booting stage, we collected and processed anthers for transverse sectioning immediately and at the flowering stage. We anatomically evaluated the effect of cold treatment on anther internal morphologies, pollen fertilities and pollen numbers in the 13 cultivars with various cold sensitivities. KEY RESULTS: We observed four types of morphological anther abnormalities at each stage. Pollen sterility was positively correlated with the frequency of undeveloped locules, but not with tapetum hypertrophy as commonly believed. In cold-sensitive cultivars grown at low temperatures, pollen sterility was more frequent than anther morphological abnormalities, and some lines showed remarkably high pollen sterility without any anther morphological alterations. Most morphological anomalies occurred only in specific areas within large and small locules. Anther length tended to shorten in response to cold treatment and was positively correlated with pollen number. One cultivar showed a considerably reduced pollen number, but fertile pollen grains under cold stress. We propose three possible relationships to explain anther structure and pollen sterility and reduction due to cold stress. CONCLUSIONS: The pollen sterility caused by cold stress at the booting stage was correlated with the frequency of entire locule-related abnormalities, which might represent a phenotypic consequence, but not a direct cause of pollen abortion. Multivalent factors might underlie the complicated relationships between anther abnormality and pollen sterility in rice.

NDR/LATS-family protein kinase genes are indispensable for embryogenesis in Arabidopsis.

NDR/LATS-family protein kinases are conserved among eukaryotes. These protein kinases in yeast and animals phosphorylate specific targets and regulate the cell cycle. Arabidopsis thaliana has eight NDR/LATS-family protein kinase genes (NDR1-8), of which NDR2, NDR4, and NDR5 are involved in regulating pollen development. However, the functions of the other NDR/LATS-family protein kinase genes in plants are unclear. Here, we show that three putative phosphorylation sites of an Arabidopsis basic leucine zipper transcription factor, VIP1, correspond to NDR/LATS-family protein kinase phosphorylation motifs and that two of these three sites are phosphorylated by NDR2, NDR3, or NDR8 in vitro. Expression of NDR1-8 was detected in various tissues. An NDR4 NDR6 NDR7 NDR8 quadruple mutation caused embryonic lethality These results suggest that different NDR/LATS-family protein kinases in plants have distinct physiological roles.

The plant nuclear lamina proteins NMCP1 and NMCP2 form a filamentous network with lateral filament associations.

Plant genomes lack genes encoding intermediate filament proteins, including lamins; however, functional lamin analogues are presumed to exist in plants. Plant-specific coiled-coil proteins, that is, nuclear matrix constituent proteins (NMCPs), are the most likely candidates as the structural elements of the nuclear lamina because they exhibit a lamin-like domain arrangement. They are exclusively localized at the nuclear periphery and have functions that are analogous to those of lamins. However, their assembly into filamentous polymers has not yet been confirmed. In this study, we examined the higher-order structure of NMCP1 and NMCP2 in Apium graveolens cells by using stimulated emission depletion microscopy combined with immunofluorescence cell labelling. Our analyses revealed that NMCP1 and NMCP2 form intricate filamentous networks, which include thick segments consisting of filament bundles, forming a dense filamentous layer extending across the nuclear periphery. Furthermore, the outermost chromatin distribution was found to be in the nucleoplasm-facing region of the nuclear lamina. Recombinant Daucus carota NMCP1 with a His-tag produced in Escherichia coli refolded into dimers and self-assembled into filaments and filament bundles. These results suggest that NMCP1 and NMCP2 organize into the nuclear lamina by forming a filamentous network with filament bundles that localize at the nuclear periphery.

The B″-family subunits of protein phosphatase 2A are necessary for in-vitro dephosphorylation of the Arabidopsis mechanosensory transcription factor VIP1.

Protein phosphatase 2A (PP2A) B″-family subunits have Ca2+-binding EF-hand motifs and can bind PP2A substrates. Arabidopsis thaliana PP2A B″-family subunits are encoded by six genes, and bind a transcription factor, VIP1. VIP1 is dephosphorylated and nuclear-localized by hypo-osmotic stress. However, whether PP2A B″-family subunits mediate the VIP1 dephosphorylation is unclear. Here, we show by yeast two-hybrid and in vitro pull down assays that Arabidopsis PP2A B″-family subunits bind Arabidopsis PP2A A (scaffold) subunits. We also show that VIP1 dephosphorylation in vitro can be induced by a PP2A B″-family subunit.

A GDSL-type esterase/lipase gene, GELP77, is necessary for pollen dissociation and fertility in Arabidopsis.

Pollen wall characteristics are dramatically changed during pollen maturation. Many genes have been identified as regulators of such changes in pollen wall characteristics, but mechanisms of such changes have not been completely understood. Here, a GDSL-type esterase/lipase gene, GELP77, is shown to regulate such changes in Arabidopsis thaliana. GELP77-deficient (gelp77) plants exhibited male sterility, and this phenotype was suppressed by introduction of a GELP77 genomic fragment. Mature pollen grains of wild-type Arabidopsis plants have an organized reticulate surface structure and are dissociated from each other. In contrast, pollen grains of gelp77 lacked such a structure and were shrunken and stuck to each other. Nuclei were not detectable in gelp77 microspores at a putative uninucleate stage, suggesting that GELP77 is required as early as this stage. In plants that have the GELP77 promoter-GELP77-GFP transgene, the GELP77-GFP fusion protein was detected in microspores, tapetal cells and middle layer cells in anthers at post-meiotic stages, whereas not anthers at pre-meiotic stages. Analysis of amino acid sequences suggests that GELP77 is phylogenetically distant from the other 104 GDSL-type esterase/lipase genes in Arabidopsis and that GELP77 orthologs are present in various plant species. Together, these results indicate that GELP77 regulates pollen wall characteristics in Arabidopsis.

A putative AGAMOUS ortholog is a candidate for the gene determining ease of dehulling in Tartary buckwheat (Fagopyrum tataricum).

MAIN CONCLUSION: Tartary buckwheat rice-type cultivars, which allow easy dehulling, lacked periclinal cell divisions that proceed underneath the epidermis in the proximity of ovary midribs in non-rice-type cultivars. The easy dehulling in these cultivars was associated with a G→A substitution in an AGAMOUS ortholog. Ease of dehulling in Tartary buckwheat (Fagopyrum tataricum) can affect the quality of its products. Tartary buckwheat cultivars that allow easy dehulling are called rice-type cultivars. The rice and non-rice hull types are determined by a single gene, but this gene is unclear. Here, we show that cells underneath the epidermis in the proximity of ovary midribs undergo periclinal cell divisions in non-rice-type cultivars but do not in a rice-type cultivar. The cells that arose from the periclinal cell divisions later underwent lignification, which should increase mechanical strength of hulls. In RNA sequencing, a partial mRNA of an AGAMOUS ortholog in Tartary buckwheat (FtAG) was found to be absent in the rice-type cultivar. Cloning of this gene revealed that this is a 42-bp deletion due to a G→A substitution at a splice acceptor site in the FtAG genomic region. In F2 progeny derived from a cross between non-rice-type and rice-type cultivars, all the rice-type plants exhibited the homozygous A/A allele at this site, whereas all the Tartary-type plants exhibited either the homozygous G/G allele or the heterozygous A/G allele. These results suggest that FtAG is a candidate for the gene that determines ease of dehulling in Tartary buckwheat. The DNA marker that we developed to distinguish the FtAG alleles can be useful in breeding Tartary buckwheat cultivars.

VIP1, a bZIP protein, interacts with the catalytic subunit of protein phosphatase 2A in Arabidopsis thaliana.

VirE2-INTERACTING PROTEIN1 (VIP1) is a basic leucine zipper protein in Arabidopsis thaliana. VIP1 changes its subcellular localization from the cytoplasm to the nucleus when cells are exposed to mechanical or hypo-osmotic stress. The nuclear localization of VIP1 is inhibited either by inhibitors of calcium signaling or by inhibitors of protein phosphatases 1, 2A and 4 (PP1, PP2A and PP4, respectively). VIP1 binds to the PP2A B"-family subunits, which have calcium-binding EF-hand motifs and which act as the regulatory, substrate-recruiting B subunit of PP2A. The VIP1 de-phosphorylation can therefore be mediated by PP2A. However, details of the PP2A-mediated de-phosphorylation of VIP1 are unclear. Here, with yeast two-hybrid assays and in-vitro pull-down assays, we show that VIP1 does not interact with the scaffolding A subunit of PP2A, but that VIP1 does interact with the catalytic C subunits. Our data raise the possibility that not only the B"-family B subunit of PP2A but also its C subunit contributes to the PP2A-mediated de-phosphorylation of VIP1.

Data of whole genome sequencing of five garden asparagus (Asparagus officinalis) individuals with the MinION nanopore sequencer.

Garden asparagus (Asparagus officinalis) is a perennial, dioecious crop. Genomic DNA samples were prepared from five A. officinalis individuals that differ in sex and phenotypes, and sequenced with the MinION nanopore sequencer. The obtained data were 1.5-5 Gb/sample, and the average read length was larger than 1.4 kb for all the samples. The resulting reads were mapped to the existing A. officinalis genome sequence. The existing A. officinalis transcript sequences were mapped to the MinION-derived reads. On the basis of these mapping results, flanking sequences of five partial gene fragments that previously had not been mapped to any region of the existing genome were determined by genomic PCR followed by Sanger sequencing. These sequences enabled to estimate the genomic positions of those five partial gene fragments. The MinION-derived data and the flanking sequences of the five gene fragments were deposited in the NCBI (National Center for Biotechnology Information) SRA (Sequence Read Archive) database and the NCBI Nucleotide database, respectively.

Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress.

VIP1 is a bZIP transcription factor in Arabidopsis thaliana. When cells are exposed to mechanical stress, VIP1 transiently accumulates in the nucleus, where it regulates the expression of its target genes and suppresses mechanical stress-induced root waving. The nuclear-cytoplasmic shuttling of VIP1 is regulated by phosphorylation and calcium-dependent signaling, but specific regulators of these processes remain to be identified. Here, inhibitors of protein phosphatase 2A (PP2A) are shown to inhibit both the mechanical stress-induced dephosphorylation and nuclear accumulation of VIP1. The PP2A B subunit, which recruits substrates of PP2A holoenzyme, is classified into B, B', B'', and B''' families. Using bimolecular fluorescence complementation, in vitro pull-down, and yeast two-hybrid assays, we show that VIP1 interacts with at least two of the six members of the Arabidopsis PP2A B''-family subunit, which have calcium-binding EF-hand motifs. VIP1AAA, a constitutively nuclear-localized VIP1 variant with substitutions in putative phosphorylation sites of VIP1, suppressed the root waving induced by VIP1-SRDX (a repression domain-fused variant of VIP1). These results support the idea that VIP1 is dephosphorylated by PP2A and that the dephosphorylation suppresses the root waving. The phosphorylation sites of VIP1 and its homologs were narrowed down by in vitro phosphorylation, yeast two-hybrid, and protein subcellular localization assays.

Development of a DNA marker for variety discrimination specific to 'Manten-Kirari' based on an NGS-RNA sequence in Tartary buckwheat (Fagopyrum tataricum).

To discriminate the trace-rutinosidase variety of Tartary buckwheat 'Manten-Kirari', we developed DNA markers based on RNA polymorphism. Specifically, we mapped 17.76 GB RNA sequences, obtained using HiSeq2000, to create 11,358 large contigs constructed de novo from 'Manten-Kirari' RNA derived from GS-FLX+ titanium. From these, we developed eight DNA markers corresponding to single- to four-nucleotide polymorphisms between 'Manten-Kirari' and 'Hokkai T8', which is representative of normal rutinosidase content varieties in Japan. Using these markers, 'Manten-Kirari' was discriminated from 'Hokkai T8' by eight markers, from major Tartary buckwheat varieties by three markers, and from common buckwheats by two markers. We also performed direct PCR from flour and dried noodle made with 'Manten-Kirari' and 'Hokkai T8'. Based on the results, the DNA markers developed are promising for discriminating 'Manten-Kirari'. This is the first study to develop a DNA marker to discriminate varieties in the Polygonaceae family including buckwheat species.

CRISPR/Cas9-Mediated Editing of Genes Encoding rgs-CaM-like Proteins in Transgenic Potato Plants.

A tobacco calmodulin-like protein, rgs-CaM, has been shown to interact with viruses in a variety of ways; it contributes to geminivirus infections but is also involved in primed immunity to the cucumber mosaic virus. Sequence similarity searches revealed several calmodulin-like proteins similar to rgs-CaM (rCML) in Arabidopsis and other Solanaceae plants, including potato (Solanum tuberosum). To analyze the functions of each rCML, mutations were introduced into potato rCMLs using the CRISPR/Cas9 system. Here, we describe our protocol of the CRISPR/Cas9-mediated targeted mutagenesis in stably transformed potato plants.

Death of female flower microsporocytes progresses independently of meiosis-like process and can be accelerated by specific transcripts in Asparagus officinalis.

Asparagus officinalis (garden asparagus) is a dioecious perennial crop, and the dioecy (i.e., sex) of A. officinalis can affect its productivity. In A. officinalis, flower anthers in female plants fail to accumulate callose around microsporocytes, fail to complete meiosis, and degenerate due to cell death. Although 13 genes have been implicated in the anther development of male and female flowers, it is unclear how these genes regulate the cell death in female flower anthers. The aim of this study was to narrow down factors involved in this process. TUNEL staining and Feulgen staining of female flower microsporocytes suggest that female microsporocytes enter a previously undetected meiosis-like process, and that the cell death occurs independently of this meiosis-like process, excluding the possibility that the cell death is caused by the cessation of meiosis. RNA sequencing with individual floral organs (tepals, pistils and stamens) revealed that several genes possibly regulating the cell death, such as metacaspase genes and a Bax inhibitor-1 gene, are differentially regulated between female and male flower anthers, and that genes involved in callose accumulation are up-regulated only in male flower anthers. These genes are likely involved in regulating the cell death in female flower anthers in A. officinalis.

B-family subunits of protein phosphatase 2A are necessary for pollen development but not for female gametophyte development in Arabidopsis.

Protein phosphatase 2A (PP2A) is a heterotrimeric protein complex conserved among eukaryotes. The B subunit of PP2A determines the substrate specificity of the PP2A holoenzyme, and is classified into the B, B', B″ and B‴ families. Arabidopsis thaliana has two isoforms of the B-family subunit (ATBA and ATBB). A double knockout of their genes is lethal, but which developmental process is primarily impaired by the double knockout is unclear. Identifying such a process helps understand PP2A-mediated signaling more deeply. Here, genetic characterization of new knockout mutants for these genes shows that they are necessary for pollen development but not for female gametophyte development. Compared to wild-type pollen grains, the mutant pollen grains exhibited lower enzyme activities, germinated less frequently on stigmas, and exhibited the aberrant numbers of sperm cell nuclei, suggesting that ATBA and ATBB play pleiotropic roles in pollen development. The amino acids stabilizing the interaction between the human PP2A A and B-family subunits are conserved in an Arabidopsis A subunit (AtPP2AA2), ATBA and ATBB. His-tagged AtPP2AA2 co-immunoprecipitated with either Myc-tagged ATBA or Myc-tagged ATBB in vitro, confirming their interactions. Proteins that regulate pollen development and that undergo dephosphorylation are likely primary targets of ATBA and ATBB.

Possible inhibition of Arabidopsis VIP1-mediated mechanosensory signaling by streptomycin.

VIP1 (VIRE2-INTERACTING PROTEIN 1) and its close homologues are Arabidopsis thaliana bZIP proteins regulating stress responses and root tropisms. They are present in the cytoplasm under steady conditions, but transiently accumulate in the nucleus when cells are exposed to mechanical stress such as hypo-osmotic stress and touch. This pattern of changes in subcellular localization is unique to VIP1 and its close homologues, and can be useful to further characterize mechanical stress signaling in plants. A recent study showed that calcium signaling regulates this pattern of subcellular localization. Here, we show that a possible calcium channel inhibitor, streptomycin, also inhibits the nuclear accumulation of VIP1. Candidates for the specific regulators of the mechanosensitive calcium signaling are further discussed.

Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana.

Background and Aims: VIP1 is a bZIP transcription factor in Arabidopsis thaliana. VIP1 and its close homologues transiently accumulate in the nucleus when cells are exposed to hypo-osmotic and/or mechanical stress. Touch-induced root bending is enhanced in transgenic plants overexpressing a repression domain-fused form of VIP1 (VIP1-SRDXox), suggesting that VIP1, possibly with its close homologues, suppresses touch-induced root bending. The aim of this study was to identify regulators of these functions of VIP1 in mechanical stress responses. Methods: Co-immunoprecipitation analysis using VIP1-GFP fusion protein expressed in Arabidopsis plants identified calmodulins as VIP1-GFP interactors. In vitro crosslink analysis was performed using a hexahistidine-tagged calmodulin and glutathione S-transferase-fused forms of VIP1 and its close homologues. Plants expressing GFP-fused forms of VIP1 and its close homologues (bZIP59 and bZIP29) were submerged in hypotonic solutions containing divalent cation chelators, EDTA and EGTA, and a potential calmodulin inhibitor, chlorpromazine, to examine their effects on the nuclear-cytoplasmic shuttling of those proteins. VIP1-SRDXox plants were grown on medium containing 40 mm CaCl2, 40 mm MgCl2 or 80 mm NaCl. MCA1 and MCA2 are mechanosensitive calcium channels, and the hypo-osmotic stress-dependent nuclear-cytoplasmic shuttling of VIP1-GFP in the mca1 mca2 double knockout mutant background was examined. Key Results: In vitro crosslink products were detected in the presence of CaCl2, but not in its absence. EDTA, EGTA and chlorpromazine all inhibited both the nuclear import and the nuclear export of VIP1-GFP, bZIP59-GFP and bZIP29-GFP. Either 40 mm CaCl2or 80 mm NaCl enhanced the VIP-SRDX-dependent root bending. The nuclear-cytoplasmic shuttling of VIP1 was observed even in the mca1 mca2 mutant. Conclusions: VIP1 and its close homologues can interact with calmodulins. Their nuclear-cytoplasmic shuttling requires neither MCA1 nor MCA2, but does require calcium signalling. Salt stress affects the VIP1-dependent regulation of root bending.