Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The beta-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85-90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.

SummaryIn higher plants, gravitropism proceeds through three sequential steps in the responding organs: perception of gravity signals, signal transduction and asymmetric cell elongation. Light and temperature also influence the gravitropic orientation of plant organs. A series of Arabidopsis shoot gravitropism (sgr) mutants has been shown to exhibit disturbed shoot gravitropism. SGR5 is functionally distinct from other SGR members in that it mediates the early events of gravitropic responses in inflorescence stems.Here, we demonstrated that SGR5 alternative splicing produces two protein variants (SGR5a and SGR5b) in modulating the gravitropic response of inflorescence stems at high temperatures. SGR5b inhibits SGR5a function by forming non-DNA-binding heterodimers.Transgenic plants overexpressing SGR5b (35S:SGR5b) exhibit reduced gravitropic growth of inflorescence stems, as observed in the SGR5-deficient sgr5-5 mutant. Interestingly, SGR5 alternative splicing is accelerated at high temperatures, resulting in the high-level accumulation of SGR5b transcripts. When plants were exposed to high temperatures, whereas gravitropic curvature was reduced in Col-0 inflorescence stems, it was uninfluenced in the inflorescence stems of 35S:SGR5b transgenic plants and sgr5-5 mutant.We propose that the thermoresponsive alternative splicing of SGR5 provides an adaptation strategy by which plants protect the shoots from hot air under high temperature stress in natural habitats.

The ATPases associated with various cellular activities (AAA) proteins are widespread in living organisms. Some of the AAA-type ATPases possess metalloprotease activities. Other members constitute the 26S proteasome complexes. In recent years, a few AAA members have been implicated in vesicle-mediated secretion, membrane fusion, cellular organelle biogenesis, and hypersensitive responses (HR) in plants. However, the physiological roles and biochemical activities of plant AAA proteins have not yet been defined at the molecular level, and regulatory mechanisms underlying their functions are largely unknown. In this study, we showed that overexpression of an Arabidopsis gene encoding a mitochondrial AAA protein, ATPase-in-Seed-Development (ASD), induces morphological and anatomical defects in seed maturation. The ASD gene is expressed at a high level during the seed maturation process and in mature seeds but is repressed rapidly in germinating seeds. Transgenic plants overexpressing the ASD gene are morphologically normal. However, seed formation is severely disrupted in the transgenic plants. The ASD gene is induced by abiotic stresses, such as low temperatures and high salinity, in an abscisic acid (ABA)-dependent manner. The ASD protein possesses ATPase activity and is localized into the mitochondria. Our observations suggest that ASD may play a role in seed maturation by influencing mitochondrial function under abiotic stress.