AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin

Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.     

A Dominant Mutation in the Light-Oxygen and Voltage2 Domain Vicinity Impairs Phototropin1 Signaling in Tomato

In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A′α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A′α helix region in phototropic signaling of tomato.Being sessile in nature, plants have developed diverse sets of sensory mechanisms, integrating external cues such as light, water, and temperature to adapt their growth and development to the ambient environment. Plants have evolved a cohort of photoreceptors such as red/far-red light-sensing phytochromes (Chen and Chory, 2011), UV-A/blue light (BL)-sensing phototropins (Christie, 2007; Holland et al., 2009; Suetsugu and Wada, 2013), cryptochromes (Yu et al., 2010; Liu et al., 2011), Zeitlupe (ZTL)/Flavin-binding, Kelch repeat, F-box protein1/light-oxygen and voltage (LOV)-kelch protein2 members of the ZTL/ADAGIO putative family of photoreceptors (Suetsugu and Wada, 2013), and UV-B light-sensing UV-B resistance8 (Heijde and Ulm, 2012), enabling them to sense nearly the full range of the solar spectrum. One of the most visually obvious photoresponses of flowering plants involves the growth and orientation of organs toward or away from light, particularly during the early stages of growth and the establishment of seedlings (Iino, 1990) and during gap-filling situations in dense canopy conditions (Ballaré, 1999) for optimizing photosynthesis and interspecies/intraspecies competition. Several studies involving the relative effectiveness of different wavelengths of the solar spectrum as well as monitoring of lateral differences in light intensity revealed that the directional growth of plants is specifically mediated by the UV-A/blue region of the visible spectrum. Molecular genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants inhibited in hypocotyl curvature toward BL revealed that, among the UV-A light-/BL-specific photoreceptors, the phototropins perceive ambient light as a cue for directional growth (Liscum and Briggs, 1995; Kagawa et al., 2001).Phototropins have been identified in several plant species, ranging from the green alga Chlamydomonas reinhardtii to higher plants (Briggs et al., 2001). To date, two members of the phototropins have been reported from higher plants, phot1 and phot2, which share sequence homology (Sakai et al., 2001). Physiological analyses with Arabidopsis mutants lacking phot1 and phot2 have revealed that, in addition to regulating the hypocotyl curvature of seedlings toward BL (Huala et al., 1997; Christie et al., 1998), phototropins also regulate a diverse range of responses in flowering plants (Christie and Murphy, 2013; Hohm et al., 2013). These responses include chloroplast movements (Sakai et al., 2001), nuclear positioning (Iwabuchi et al., 2007), stomatal opening (Kinoshita et al., 2001), sun tracking (Inoue et al., 2008b), leaf expansion (Ohgishi et al., 2004), leaf movements (Inoue et al., 2005), leaf photomorphogenesis (Kozuka et al., 2011), leaf flattening (Sakamoto and Briggs, 2002), and the rapid inhibition of the growth of etiolated hypocotyls (Folta and Spalding, 2001).While both phot1 and phot2 overlap in function in regulating phototropism, chloroplast accumulation, leaf expansion, and stomatal opening, they also exhibit differential photosensitivity to BL, where phot1 is more sensitive to low-fluence BL than phot2. Both phot1 and phot2 redundantly regulate the chloroplast accumulation toward low-fluence BL, and phot2 exclusively regulates the chloroplast avoidance from high-fluence BL (Jarillo et al., 2001; Kagawa et al., 2001), while phot1 solely mediates the rapid inhibition of the elongation of etiolated hypocotyls (Folta and Spalding, 2001). Analysis of mutants downstream of blue light perception by phototropins revealed that the phototropin signaling branches out at an early step, and phot1 and phot2 trigger distinct photoresponses recruiting multiple signaling partners (Christie and Murphy, 2013; Hohm et al., 2013).Molecular characterizations have shown that phototropins are plasma membrane-associated Ser/Thr kinases containing a photosensory domain (Briggs and Christie, 2002) in the N-terminal region composed of two LOV domains (LOV1 and LOV2) and the kinase domain at the C-terminal end. The LOV1 and LOV2 domains bind the FMN as chromophore and are responsible for BL sensing by phototropin. Although phototropins characteristically possess two LOV domains, the photoregulation of phototropin activity is predominantly mediated by LOV2 (Christie, 2007). The exposure to BL also causes adduct formation between the FMN and the Cys residue in LOV domains and leads to the phosphorylation of phototropin, which is believed to be the primary step in the transmission of phototropic signals (Christie et al., 1998; Sakai et al., 2000). To decipher the functions of different domains of phototropins, many different substitution mutants of phototropins have been generated, which have enabled the elucidation of the functional significance of the different domains (Matsuoka and Tokutomi, 2005; Jones et al., 2007; Kong et al., 2007; Inoue et al., 2008a). Inoue et al. (2008a) showed that the BL-induced autophosphorylation of Ser-851 in the C-terminal kinase domain of phototropin is the primary step for initiating stomatal opening, phototropism, chloroplast accumulation, and leaf flattening. Mutational studies also revealed that the photosensory N-terminal domain of phototropin acts as a kinase inhibitor, where the LOV2 domain inhibits the activity of kinase domain by binding to it, and BL exposure is required for the dissociation of the LOV2 domain, enabling phosphorylation of the kinase domain (Matsuoka and Tokutomi, 2005; Jones et al., 2007).While our current understanding of phototropism has been greatly facilitated by the isolation of phototropins and their signaling mutants, the phot mutants identified to date are loss-of-function alleles. The lack of dominant-negative alleles or alleles with increased sensitivity to phototropic stimulus has hindered exploration into the roles of different domains of phot proteins in regulating phototropic signaling. In addition, the dearth of mutants defective in phototropin or phototropin-mediated responses has been a major bottleneck in furthering our understanding of the function of phototropins in crop species. Although phototropin homologs have been identified from a variety of crop species, including oat (Avena sativa; Zacherl et al., 1998), rice (Oryza sativa; Kanegae et al., 2000), and tomato (Solanum lycopersicum; Sharma et al., 2007; Sharma and Sharma, 2007), only the coleoptile phototropism1 mutant of rice has been isolated, which is defective in BL phototropism (Haga et al., 2005).Here, we report that in a mutant screen for nonphototropic seedlings under continuous BL, we recovered a strong dominant-negative mutation of phot1. The dominant-negative mutations are useful to elucidate redundant functions, as mutant protein in addition to suppressing its own functions can also suppress the function of its partners. The characterization of this new phot1 mutant revealed that the dominant activity is caused by the substitution of an Arg residue located in the A′α helix in the Hinge1 region between the LOV1 and LOV2 domains. Our study shows the functional importance of the A′α helix (Halavaty and Moffat, 2007) in regulating phot1-mediated signaling in tomato.     

Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.     

The root as a drill: An ethylene-auxin interaction facilitates root penetration in soil

Plant roots forage the soil for water and nutrients and overcome the soil’s physical compactness. Roots are endowed with a mechanism that allows them to penetrate and grow in dense media such as soil. However, the molecular mechanisms underlying this process are still poorly understood. The nature of the media in which roots grow adds to the difficulty to in situ analyze the mechanisms underlying root penetration. Inhibition of ethylene perception by application of 1-methyl cyclopropene (1-MCP) to tomato seedlings nearly abolished the root penetration in Soilrite. The reversal of this process by auxin indicated operation of an auxin-ethylene signaling pathway in the regulation of root penetration. The tomato pct1–2 mutant that exhibits an enhanced polar transport of auxin required higher doses of 1-MCP to inhibit root penetration, indicating a pivotal role of auxin transport in this process. In this update we provide a brief review of our current understanding of molecular processes underlying root penetration in higher plants.     

Generation of genetically stable transformants by <Emphasis Type="Italic">Agrobacterium</Emphasis> using tomato floral buds

Tomato (Solanum lycopersicum) is a model crop plant for the study of fruit ripening and disease resistance. Here we present a systemic study on in planta transformation of tomato with Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA1303 binary vector bearing HPTII as a plant selectable marker and mGFP/GUS fusion as the reporter gene. We attempted the transformation of tomato at different developmental stages viz. during seed germination, seedling growth, and floral bud development. The imbibition of seeds with Agrobacterium suspension led to seed mortality. The vacuum infiltration of seedlings with Agrobacterium suspension led to sterility in surviving plants. Successful transformation could be achieved either by dipping of developing floral buds in the Agrobacterium suspension or by injecting Agrobacterium into the floral buds. Most floral buds subjected to dip as well as to injection either aborted or had arrested development. The pollination of surviving floral buds with pollen from wild-type plants yielded fruits bearing seeds. A transformation efficiency of 0.25–0.50% was obtained on floral dips/floral injections. Transgenic plants were selected by screening seedlings for hygromycin resistance. The presence of the transgene in genomic DNA was confirmed by Southern blot analysis and expression of the reporter gene up to the T₄ generation. The amenability of tomato for in planta transformation simplifies the generation of transgenic tomato plants obviating intervening tissue culture.     

The polycotyledon mutant of tomato shows enhanced polar auxin transport

The polycotyledon mutant of tomato (Lycopersicon esculentum L. cv Ailsa Craig) showed altered development during embryogenesis and during vegetative and reproductive phases. The phenotype was pleiotropic and included the formation of extra cotyledons, changes in leaf shape, increased number of flowers (indeterminacy) with abnormal floral organs, the formation of epiphyllous structures, and altered gravitropism. The earliest defects were observed at the transition from the globular to the heart stage of embryogenesis with the formation of multiple cotyledons. Epidermal cells in the mutant embryo were smaller and less expanded compared with wild type. Examination of polar auxin transport (PAT) showed a striking enhancement in the case of the mutant. Increase in PAT did not appear to be caused by a decrease in flavonoids because the mutant had normal flavonoid levels. Application of 2,3,5-triiodobenzoic acid, an inhibitor of polar transport of auxin, rescued postgermination phenotypes of young seedlings. Our analysis reveals a level of control that negatively regulates PAT in tomato and its contribution to plant development and organogenesis.     

Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade

The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two B^og (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the “Beta type” promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening.     

Light modulates the root tip excision induced lateral root formation in tomato

During plant growth and development, root tip performs multifarious functions integrating diverse external and internal stimuli to regulate root elongation and architecture. It is believed that a signal originating from root tip inhibits lateral root formation (LRF). The excision of root tip induced LRF in tomato seedlings associated with accumulation of auxin in pericycle founder cells. The excision of cotyledons slightly reduced LRF, whereas severing shoot from root completely abolished LRF. Exogenous ethylene application did not alter LRF. The response was modulated by light with higher LRF in seedlings exposed to light. Our results indicate that light plays a role in LRF in seedlings by likely modulating shoot derived auxin.