SummaryPhloem-mobile signals play a major role in plant nutrition, development and communication. In the latter context, phloem-mobile RNAs have been associated with signalling between plant tissues. In this study, we focused on the identification of transcripts in the shoot phloem of the model plant Arabidopsis thaliana. To isolate transcripts expressed in phloem parenchyma cells and in companion cell-sieve element complexes, we used laser microdissection coupled to laser pressure catapulting (LMPC). Mobile transcripts in sieve elements were isolated from leaf phloem exudates. After optimization of sampling and fixation, RNA of high quality was isolated from both sources. The modifications to the RNA amplification procedure described here were well suited to production of RNA of sufficient yield and quality for microarray experiments. Microarrays hybridized with LMPC-derived phloem tissue or phloem sap RNA allowed differentiation between phloem-expressed and mobile transcript species. Using this set of phloem transcripts and comparing them with microarrays derived from databases of light, hormone and nutrient treatment experiments, we identified phloem-derived RNAs as mobile, potential long-distance signals. Our dataset thus provides a search criterion for phloem-based signals hidden in the complex datasets of microarray experiments. The availability of these comprehensive phloem transcript profiles will facilitate reverse-genetic studies and forward-genetic screens for phloem and longdistance RNA signalling mutants.

Nonspecific lipid transfer proteins reversibly bind different types of lipid molecules in a hydrophobic cavity. They facilitate phospholipid transfer between membranes in vitro, play a role in cuticle and possibly in suberin formation, and might be involved in plant pathogen defense signaling. This study focuses on the role of the lipid transfer protein AtLTPI-4 in crown gall development. Arabidopsis (Arabidopsis thaliana) crown gall tumors, which develop upon infection with the virulent Agrobacterium tumefaciens strain C58, highly expressed AtLTPI-4. Crown galls of the atltpI-4 loss-of-function mutant were much smaller compared with those of wild-type plants. The gene expression pattern and localization of the protein to the plasma membrane pointed to a function of AtLTPI-4 in cell wall suberization. Since Arabidopsis crown galls are covered by a suberin-containing periderm instead of a cuticle, we analyzed the suberin composition of crown galls and found a reduction in the amounts of long-chain fatty acids (C 18:0 ) in the atltpI-4 mutant. To demonstrate the impact of AtLtpI-4 on extracellular lipid composition, we expressed the protein in Arabidopsis epidermis cells. This led to a significant increase in the very-long-chain fatty acids C 24 and C 26 in the cuticular wax fraction. Homology modeling and lipid-protein-overlay assays showed that AtLtpI-4 protein can bind these very-long-chain fatty acids. Thus, AtLtpI-4 protein may facilitate the transfer of long-chain as well as very-long-chain fatty acids into the apoplast, depending on the cell type in which it is expressed. In crown galls, which endogenously express AtLtpI-4, it is involved in suberin formation.

Agrobacterium tumefaciens-derived crown galls of Arabidopsis (Arabidopsis thaliana) contain elevated levels of unsaturated fatty acids and strongly express two fatty acid desaturase genes, v3 FATTY ACID DESATURASE3 (FAD3) and STEAROYL-ACYL CARRIER PROTEIN D 9-DESATURASE6 (SAD6). The fad3-2 mutant with impaired a-linolenic acid synthesis developed significantly smaller crown galls under normal, but not under high, relative humidity. This strongly suggests that FAD3 plays a role in increasing drought stress tolerance of crown galls. SAD6 is a member of the SAD family of as yet unknown function. Expression of the SAD6 gene is limited to hypoxia, a physiological condition found in crown galls. As no sad6 mutant exists and to link the function of SAD6 with fatty acid desaturation in crown galls, the lipid pattern was analyzed of plants with constitutive SAD6 overexpression (SAD6-OE). SAD6-OE plants contained lower stearic acid and higher oleic acid levels, which upon reduction of SAD6 overexpression by RNA interference (SAD6-OE-RNAi) regained wild-type-like levels. The development of crown galls was not affected either in SAD6-OE or SAD6-OE-RNAi or by RNA interference in crown galls. Since biochemical analysis of SAD6 in yeast (Saccharomyces cerevisiae) and Escherichia coli failed, SAD6 was ectopically expressed in the background of the well-known suppressor of salicylic acid-insensitive2 (ssi2-2) mutant to confirm the desaturase function of SAD6. All known ssi2-2 phenotypes were rescued, including the high stearic acid level. Thus, our findings suggest that SAD6 functions as a D 9-desaturase, and together with FAD3 it increases the levels of unsaturated fatty acids in crown galls under hypoxia and drought stress conditions.

This rapid protocol allows the extraction of chloroplast enriched proteins fromNicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined with western-blot analysis.