HEXOKINASE1 (HXK1) from Arabidopsis (Arabidopsis thaliana) has dual roles in glucose (Glc) signaling and in Glc phosphorylation. The cellular context, though, for HXK1 function in either process is not well understood. Here we have shown that within normal experimental detection limits, AtHXK1 is localized continuously to mitochondria. Two mitochondrial porin proteins were identified as capable of binding to overexpressed HXK1 protein, both in vivo and in vitro. We also found that AtHXK1 can be associated with its structural homolog, F-actin, based on their coimmunoprecipitation from transgenic plants that overexpress HXK1-FLAG or from transient expression assays, and based on their localization in leaf cells after cryofixation. This association might be functionally important because Glc signaling in protoplast transient expression assays is compromised by disruption of F-actin. We also demonstrate that Glc treatment of Arabidopsis seedlings rapidly and reversibly disrupts fine mesh actin filaments. The possible roles of actin in HXK-dependent Glc signaling are discussed.

Plant growth and development is regulated by complex interactions among different hormonal, developmental and environmental signalling pathways. Isolation of mutants in these processes is a powerful approach to dissect unknown mechanisms in regulatory networks. The plant hormones abscisic acid (ABA) and auxin are involved in vegetative, developmental and environmental growth responses, including cell division and elongation, vascular tissue differentiation and stress adaptation. The uidA (beta-glucuronidase; GUS) reporter gene driven by the carrot (Daucus carota) late embryogenesis-abundantDc3 promoter in transgenic Arabidopsis thaliana seedlings is ABA-inducible in the root zone of elongation and vasculature. We show here that the ABA-insensitive2-1 mutation (abi2) reduces ABA-inducible Dc3-GUS expression in these root tissues. Dc3-GUS expression is also induced in root cortex cells by indole-3-acetic acid. We mutagenized, with ethyl methane sulfonate, 5100 M1 abi2/abi2 homozygous plants of a line that carries two independent Dc3-GUS reporter genes and screened M2 clonal lines for ABA-inducible Dc3-GUS expression in roots. We isolated two novel single-gene nuclear mutants, harlequin (hlq) and short blue root (sbr), that ectopically express Dc3-GUS in roots and have pleiotropic effects on morphogenesis. The hlq mutant expresses Dc3-GUS in a checkered pattern in epidermis of roots and hypocotyls, accumulates callose and has deformed and collapsed epidermal cells and abnormal and reduced root hairs and leaf trichomes. It (hlq) is also dwarfed, skotomorphogenic and sterile. The sbr mutant is a seedling-lethal dwarf that over-expresses Dc3-GUS in the root and has radially swollen epidermal cells in the root and hypocotyl, supernumerary cell number in the root cortex and epidermis, abnormal vasculature, and abnormal epidermal cell patterning in cotyledons and leaves. It (sbr) also exhibits a semidominant root phenotype of reduced growth and lateral root initiation. The hlq and sbr mutants are not rescued by exogenous application of plant growth regulators. The hlq and sbr mutants do not require the abi2-1 mutant gene for their phenotypes and map to chromosome III and I, respectively. Further characterization of the hlq and sbr phenotypes and genes may provide insights into the relationship of hormone- and stress-regulated gene expression to morphogenesis and plant growth.

Glucose functions in plants both as a metabolic resource as well as a hormone that regulates expression of many genes. Arabidopsis hexokinase1 (HXK1) is the best understood plant glucose sensor/ transducer, yet we are only now appreciating the cellular complexity of its signaling functions. We have recently shown that one of the earliest detectable responses to plant glucose treatments are extensive alterations of cellular F-actin. Interestingly, AtHXK1 is predominantly located on mitochondria, yet also can interact with actin. A normal functioning actin cytoskeleton is required for HXK1 to act as an effector in glucose signaling assays. We have suggested that HXK1 might alter F-actin dynamics and thereby influence the formation and/or stabilization of cytoskeleton-bound polysomes. In this Addendum, we have extended our initial observations on the subcellular targeting of HXK1 and its interaction with F-actin. We then further consider the cellular context in which HXK1 might regulate gene expression.

SummaryThe harlequin mutant encodes subunit B of plant-specific topoisomerase VI. Transcriptomics showed synergistic effects on expression of adjacent genes, suggesting a broader role in chromatin remodelling during development and stress responses.

Doubled haploid (DH) technology remarkably accelerates the crop breeding by obtaining homozygous lines in a single generation. The present study was targeted in generating haploid plants through androgenesis. Anthers from immature flower buds of six tomato genotypes viz., LE-1230, LE-1236, LE-1256 TLCV 2, PKM 1 and TNAU tomato hybrid CO 3 were used for induction of haploids. A preliminary study based on callus induction frequency (CIF), more than 5% was helpful in short listing flower bud size, pre-treatments and growth regulator combinations. Subsequently, anthers from two different sized flower buds (4 and 6 mm length), dissected either from fresh or pre-treated flower buds (2 and 5 days in dark at 4 °C or gamma irradiated) were inoculated in MS medium fortified with different growth regulators for callus induction. Among the genotypes, TLCV 2 had recorded the maximum CIF (38.80%) from anthers of 4 mm long flower buds followed by TNAU tomato hybrid CO 3 (34.70%). Throughout the study, anthers from 4 mm long flower buds responded the best for callus induction. Among the pre-treatments, anthers from gamma irradiated flower buds recorded the highest CIF (31.90%) when compared to others. Cold shock (4 °C) in dark to flower buds for 2 days had improved the CIF of anthers when compared to fresh in LE 1230, LE 1238, TLCV2 and TNAU tomato hybrid CO 3, but when the cold shock was increased to 5 days, invariably there was a reduction in CIF in all the six genotypes. TA 8 (MS + 2iP (0.5 mg L-1) + NAA (0.5 mg L-1)) medium was found to be the best for maximum CIF in LE 1230 and PKM1, TA1 (MS + 2iP (1.0 mg L-1) + IAA (2.0 mg L-1)) in LE 1238, LE 1256 and TNAU tomato hybrid CO 3 and TA7 (MS + 2iP (0.5 mg L-1) + Kinetin (1.5 mg L-1) + NAA (1.0 mg L-1)) for TLCV 2 genotypes. The callus induced was sub cultured at monthly intervals in the same medium for proliferation and later transferred to regeneration medium. A good number of shoots got regenerated only from anther calli of TNAU hybrid CO 3 that was sub cultured in MS medium fortified with Zeatin (0.5 mg L-1). The clumps of shoots induced were separated and inoculated in MS medium supplemented with GA3 (0.5 mg L-1) for shoot elongation. After 4-6 weeks, the elongated shoots were transferred to half strength MS medium enhanced with IBA (1 mg L-1). Profuse rooting from the base of the shoot was noticed in 4-5 weeks. The stomatal count with leaves from the diploid plants and in vitro plants observed were 3-4 and 1 respectively indicating the haploidy nature of in vitro plants.