The Arabidopsis thaliana histone H2A gene HTA1 is essential for efficient transformation of Arabidopsis roots by Agrobacterium tumefaciens. Disruption of this gene in the rat5 mutant results in decreased transformation. In Arabidopsis, histone H2A proteins are encoded by a 13-member gene family. RNA encoded by these genes accumulates to differing levels in roots and whole plants; HTA1 transcripts accumulate to levels up to 1000-fold lower than do transcripts of other HTA genes. We examined the extent to which other HTA genes or cDNAs could compensate for loss of HTA1 activity when overexpressed in rat5 mutant plants. Overexpression of all tested HTA cDNAs restored transformation competence to the rat5 mutant. However, only the HTA1 gene, but not other HTA genes, could phenotypically complement rat5 mutant plants when expressed from their native promoters. Expression analysis of HTA promoters indicated that they had distinct but somewhat overlapping patterns of expression in mature plants. However, only the HTA1 promoter was induced by wounding or by Agrobacterium infection of root segments. Our data suggest that, with respect to Agrobacterium-mediated transformation, all tested histone H2A proteins are functionally redundant. However, this functional redundancy is not normally evidenced because of the different expression patterns of the HTA genes.

SummaryTransformation of plant cells by Agrobacterium tumefaciens involves both bacterial virulence proteins and host proteins. We have previously shown that the Arabidopsis thaliana gene H2A-1 (RAT5), which encodes histone H2A-1, is involved in T-DNA integration into the plant genome. Mutation of RAT5 results in a severely decreased frequency of transformation, whereas overexpression of RAT5 enhances the transformation frequency (Mysore et al., 2000b). We show here that the expression pattern of RAT5 correlates with plant root cells most susceptible to transformation. As opposed to a cyclin-GUS fusion gene whose expression is limited to meristematic tissues, the H2A-1 gene is expressed in many non-dividing cells. Under normal circumstances, the H2A-1 gene is expressed in the elongation zone of the root, the region that is most susceptible to Agrobacterium transformation. In addition, when roots are incubated on medium containing phytohormones, a concomitant shift in H2A-1 expression and transformation susceptibility to the root base is observed. Inoculation of root segments with a transfer-competent, but not a transformation-deficient Agrobacterium strain induces H2A-1 expression. Furthermore, pre-treatment of Arabidopsis root segments with phytohormones both induces H2A-1 expression and increases the frequency of Agrobacterium transformation. Our results suggest that the expression of the H2A-1 gene is both a marker for, and a predictor of, plant cells most susceptible to Agrobacterium transformation.

Agrobacterium-mediated transformation is the most widely used technique for generating transgenic plants. However, many crops remain recalcitrant. We found that an Arabidopsis myb family transcription factor (MTF1) inhibited plant transformation susceptibility. Mutating MTF1 increased attachment of several Agrobacterium strains to roots and increased both stable and transient transformation in both susceptible and transformation-resistant Arabidopsis ecotypes. Cytokinins from Agrobacterium tumefaciens decreased the expression of MTF1 through activation of the cytokinin response regulator ARR3. Mutating AHK3 and AHK4, genes that encode cytokinin-responsive kinases, increased the expression of MTF1 and impaired plant transformation. Mutant mtf1 plants also had increased expression of AT14A, which encodes a putative transmembrane receptor for cell adhesion molecules. Plants overexpressing AT14A exhibited increased susceptibility to transformation, whereas at14a mutant plants exhibited decreased attachment of bacteria to roots and decreased transformation, suggesting that AT14A may serve as an anchor point for Agrobacteria. Thus, by promoting bacterial attachment and transformation of resistant plants and increasing such processes in susceptible plants, treating roots with cytokinins may help engineer crops with improved features or yield.

Recently, a new member of the ABC transporter superfamily of Arabidopsis, AtMRP5, was identified and characterized. In the present work, we found that AtMRP5 can bind specifically to sulfonurea when it is expressed in HEK293 cells. We also present evidence for a new role of AtMRP5 in the salt stress response of Arabidopsis. We used reverse genetics to identify an Arabidopsis mutant (atmrp5-2) in which the AtMRP5 gene was disrupted by transferred DNA insertion. In root-bending assays using Murashige and Skoog medium supplemented with 100 mm NaCl, root growth of atmrp5-2 was substantially inhibited in contrast to the almost normal growth of wild-type seedlings. This hypersensitive response of the atmrp5-2 mutant was not observed during mannitol treatment. The root growth of the wild-type plant grown in Murashige and Skoog medium supplemented with the MRP inhibitor glibenclamide and NaCl was inhibited to a very similar extent as the root growth of atmrp5-2 grown in NaCl alone. The Na ϩ -dependent reduction of root growth of the wild-type plant in the presence of glibenclamide was partially restored by diazoxide, a known K ϩ channel opener that reverses the inhibitory effects of sulfonylureas in animal cells. Moreover, the atmrp5-2 mutant was defective in 86 Rb ϩ uptake. When seedlings were treated with 100 mm NaCl, atmrp5-2 seedlings accumulated less K ϩ and more Na ϩ than those of the wild type. These observations suggest that AtMRP5 is a putative sulfonylurea receptor that is involved in K ϩ homeostasis and, thus, also participates in the NaCl stress response.The ATP-binding cassette (ABC) transporter superfamily is the largest known membrane transporter protein family, and its members are capable of a multitude of transport functions (Higgins, 1992(Higgins, , 1995. These proteins are highly interesting because of their involvement in numerous pathologies, such as cystic fibrosis, diabetes, and multidrug resistance (Demolombe and Escande, 1996). In animals, two ABC proteins are directly involved in regulating ion channels in the plasma membrane, specifically ATPsensitive potassium channels (K ATP channels) and the cystic fibrosis transmembrane conductance regulator (CFTR). K ATP channels were initially identified in the heart (Noma, 1983) and are complexes composed of an inward rectifier K ϩ channel and an ABC protein, the sulfonylurea receptor (Babenko et al., 1998; Miki et al., 1999). K ATP channels are highly specific for K ϩ and are inhibited by micromolar concentrations of intracellular ATP. CFTR is a chloride channel expressed by a variety of secreting epithelial cells that is regulated by cAMP-dependent phosphorylation and ATP (Anderson et al., 1991; Bear et al., 1992). These channels serve to link the electrical activity of cell membranes with cellular metabolism.The presence of ABC proteins in plants was established by the cloning of several genes encoding members of this group in Arabidopsis and other species (Dudler and Hertig, 1992; Smart and Fleming, 1996; Davies et al., 1997; Lu et al., 1997 Lu...

Agrobacterium tumefaciens caused tissue browning leading to subsequent cell death in plant transformation and novel anti-oxidative compounds enhanced Agrobacterium -mediated plant transformation by mitigating oxidative stress. Browning and death of cells transformed with Agrobacterium tumefaciens is a long-standing and high impact problem in plant transformation and the agricultural biotechnology industry, severely limiting the production of transgenic plants. Using our tomato cv. MicroTom transformation system, we demonstrated that Agrobacterium caused tissue browning (TB) leading to subsequent cell death by our correlation study. Without an antioxidant (lipoic acid, LA) TB was severe and associated with high levels of GUS transient expression and low stable transformation frequency (STF). LA addition shifted the curve in that most TB was intermediate and associated with the highest levels of GUS transient expression and STF. We evaluated 18 novel anti-oxidative compounds for their potential to enhance Agrobacterium-mediated transformation, by screening for TB reduction and monitoring GUS transient expression. Promising compounds were further evaluated for their effect on MicroTom and soybean STF. Among twelve non-antioxidant compounds, seven and five significantly (P < 0.05) reduced TB and increased STF, respectively. Among six antioxidants four of them significantly reduced TB and five of them significantly increased STF. The most efficient compound found to increase STF was melatonin (MEL, an antioxidant). Optimal concentrations and stages to use MEL in transformation were determined, and Southern blot analysis showed that T-DNA integration was not affected by MEL. The ability of diverse compounds with different anti-oxidative mechanisms can reduce Agrobacterium-mediated TB and increase STF, strongly supporting that oxidative stress is an important limiting factor in Agrobacterium-mediated transformation and the limiting factor can be controlled by these compounds at different levels.