Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood.

Transgenic hybrid aspen trees with increased gibberellin (GA) concentrations suggest that GA acts in parallel with FLOWERING LOCUS T2 to control shoot elongation.

Bioactive gibberellins (GAs) have been implicated in short day (SD)-induced growth cessation in Populus, because exogenous applications of bioactive GAs to hybrid aspens (Populus tremula × tremuloides) under SD conditions delay growth cessation. However, this effect diminishes with time, suggesting that plants may cease growth following exposure to SDs due to a reduction in sensitivity to GAs. In order to validate and further explore the role of GAs in growth cessation, we perturbed GA biosynthesis or signalling in hybrid aspen plants by overexpressing AtGA20ox1, AtGA2ox2 and PttGID1.3 (encoding GA biosynthesis enzymes and a GA receptor). We found trees with elevated concentrations of bioactive GA, due to overexpression of AtGA20ox1, continued to grow in SD conditions and were insensitive to the level of FLOWERING LOCUS T2 (FT2) expression. As transgenic plants overexpressing the PttGID1.3 GA receptor responded in a wild-type (WT) manner to SD conditions, this insensitivity did not result from limited receptor availability. As high concentrations of bioactive GA during SD conditions were sufficient to sustain shoot elongation growth in hybrid aspen trees, independent of FT2 expression levels, we conclude elongation growth in trees is regulated by both GA- and long day-responsive pathways, similar to the regulation of flowering in Arabidopsis thaliana.

Gibberellins inhibit adventitious rooting in hybrid aspen and Arabidopsis by affecting auxin transport.

Knowledge of processes involved in adventitious rooting is important to improve both fundamental understanding of plant physiology and the propagation of numerous plants. Hybrid aspen (Populus tremula × tremuloïdes) plants overexpressing a key gibberellin (GA) biosynthesis gene (AtGA20ox1) grow rapidly but have poor rooting efficiency, which restricts their clonal propagation. Therefore, we investigated the molecular basis of adventitious rooting in Populus and the model plant Arabidopsis. The production of adventitious roots (ARs) in tree cuttings is initiated from the basal stem region, and involves the interplay of several endogenous and exogenous factors. The roles of several hormones in this process have been characterized, but the effects of GAs have not been fully investigated. Here, we show that a GA treatment negatively affects the numbers of ARs produced by wild-type hybrid aspen cuttings. Furthermore, both hybrid aspen plants and intact Arabidopsis seedlings overexpressing AtGA20ox1, PttGID1.1 or PttGID1.3 genes (with a 35S promoter) produce few ARs, although ARs develop from the basal stem region of hybrid aspen and the hypocotyl of Arabidopsis. In Arabidopsis, auxin and strigolactones are known to affect AR formation. Our data show that the inhibitory effect of GA treatment on adventitious rooting is not mediated by perturbation of the auxin signalling pathway, or of the strigolactone biosynthetic and signalling pathways. Instead, GAs appear to act by perturbing polar auxin transport, in particular auxin efflux in hybrid aspen, and both efflux and influx in Arabidopsis.

Proper gibberellin localization in vascular tissue is required to control auxin-dependent leaf development and bud outgrowth in hybrid aspen.

Bioactive gibberellins (GAs) are involved in many developmental aspects in the life cycle of plants, acting either directly or through interaction with other hormones. One way to study the role of GA in specific mechanisms is to modify the levels of bioactive GA in specific tissues. We increased GA catabolism in different parts of the vascular tissue by overexpressing two different GA 2-oxidase genes that encode oxidases with affinity for C20- or C19-GA. We show that, irrespective of their localization in the vascular tissue, the expression of different members of this gene family leads to similar modifications in the primary and secondary growth of the stem of hybrid aspen. We also show that the precise localization of bioactive GA downregulation is important for the proper control of other developmental aspects, namely leaf shape and bud dormancy. Expression under the control of one of the studied promoters significantly affected both the shape of the leaves and the number of sylleptic branches. These phenotypic defects were correlated with alterations in the levels and repartitioning of auxins. We conclude that a precise localization of bioactive GA in the vasculature of the apex is necessary for the normal development of the plant through the effect of GAs on auxin transport.

Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references.

Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative 'housekeeping' genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.

Analyses of GA20ox- and GID1-over-expressing aspen suggest that gibberellins play two distinct roles in wood formation.

Gibberellins (GAs) are involved in many aspects of plant development, including shoot growth, flowering and wood formation. Increased levels of bioactive GAs are known to induce xylogenesis and xylem fiber elongation in aspen. However, there is currently little information on the response pathway(s) that mediate GA effects on wood formation. Here we characterize an important element of the GA pathway in hybrid aspen: the GA receptor, GID1. Four orthologs of GID1 were identified in Populus tremula x P. tremuloides (PttGID1.1-1.4). These were functional when expressed in Arabidopsis thaliana, and appear to present a degree of sub-functionalization in hybrid aspen. PttGID1.1 and PttGID1.3 were over-expressed in independent lines of hybrid aspen using either the 35S promoter or a xylem-specific promoter (LMX5). The 35S:PttGID1 over-expressors shared several phenotypic traits previously described in 35S:AtGA20ox1 over-expressors, including rapid growth, increased elongation, and increased xylogenesis. However, their xylem fibers were not elongated, unlike those of 35S:AtGA20ox1 plants. Similar differences in the xylem fiber phenotype were observed when PttGID1.1, PttGID1.3 or AtGA20ox1 were expressed under the control of the LMX5 promoter, suggesting either that PttGID1.1 and PttGID1.3 play no role in fiber elongation or that GA homeostasis is strongly controlled when GA signaling is altered. Our data suggest that GAs are required in two distinct wood-formation processes that have tissue-specific signaling pathways: xylogenesis, as mediated by GA signaling in the cambium, and fiber elongation in the developing xylem.

The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.

Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.

Chemical and histochemical analysis of 'Quatre Saisons Blanc Mousseux', a Moss Rose of the Rosa x damascena group.

BACKGROUND AND AIMS: Moss roses are old garden roses covered with a mossy growth on flower pedicel and calyx. This moss releases a pine-scented oleoresin that is very sticky and odoriferous. Rosa x centifolia 'muscosa' was the first moss rose to be obtained by bud-mutation but, interestingly, R. x damascena 'Quatre Saisons Blanc Mousseux' was the first repeat-blooming cultivar, thus interesting breeders. In the present study, the anatomy of these sports (i.e. bud-mutations) is characterized and the volatile organic compounds (VOCs) produced by the moss versus the petals are identified. They are compared between the two lines and their respective parents. METHODS: Anatomy of the moss is studied by environmental scanning electron microscopy and histochemical light microscopy. Sudan Red IV and Fluorol Yellow 088 are used to detect lipids, and 1-naphthol reaction with N,N-dimethyl-p-phenylenediamine to detect terpenes (Nadi reaction). Head-space or solid/liquid extraction followed by gas chromatography and mass spectrometry are used to identify VOCs in moss, trichomes and petals. KEY RESULTS: Moss of the two cultivars has the same structure with trichomes on other trichomes but not exactly the same VOCs. These VOCs are specific to the moss, with lots of terpenes. An identical VOC composition is found in leaves but not in petals. They are nearly the same in the moss mutants and in the respective wild types. CONCLUSIONS: Sepals of moss roses and their parents have a specific VOC pattern, different from that of the petals. The moss corresponds to a heterochronic mutation with trichomes developing on other trichomes. Such a mutation has probably appeared twice and independently in the two lines.