Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts.

FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis.

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506-508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4-17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways.

Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis.

The whole genome expression pattern of Arabidopsis in response to the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated using the Affymetrix ATH1-121501 array. Arabidopsis plants were grown in vitro and were exposed to 1 mM 2,4-D for 1 h, after which time gene transcription levels were measured. In response to the treatment 148 genes showed increased levels of transcription and concurrently 85 genes showed decreased levels of transcript. Genes which showed significant change in transcription levels belonged to the following functional categories: transcription, metabolism, cellular communication and signal transduction, subcellular localisation, transport facilitation, protein fate, protein with binding function or cofactor requirement and regulation of/interaction with cellular environment. Interestingly 25.3% of the genes regulated by the treatment could not be classified into a known functional category. The data obtained from these experiments were used to assess the current model of auxinic herbicide action and indicated that 2,4-D not only modulates the expression of auxin, ethylene and abscisic acid (ABA) pathways but also regulates a wide variety of other cellular functions.