A broad spectrum of host plant responses to the actions of the gall midge: case study of Robinia pseudoacacia L. and Obolodiplosis robiniae (Haldeman).

This study aims to provide insights into plant-insect interaction during the formation and development of open gall structure on the leaves of Robinia pseudoacacia during gall formation by Obolodiplosis robiniae. This was the first time such far-reaching studies were performed at a biochemical and anatomical level. The gall wall is created from a few thick cells covered with epidermis. This parenchymatous nutritive tissue is rich in starch. Sclerenchyma only occurs around the vascular bundles as a result of the lignification of the parenchyma of the bundle sheaths. The level of reactive oxygen species (ROS) in the new structure was reduced and catalase activity was inhibited, which suggests another pathway of ROS decomposition - e.g. by ascorbate or glutathione peroxidase. The gall structure was combined with an increasing level of protein and non-protein thiols. Phenols seems to be a good protective factor; whose level was lower in infected leaflets. Levels of MUFA (monosaturated fatty acids) and SFA (saturated fatty acids) rose, probably as source of food for insects. The amount of fatty acid is positively correlated with the plant response. We detected that non infected leaflets produced C6:0 (hexanoic acid) and C8:0 (octanoic acid) fatty acids connected with odor. Changes in gall color as they develop are connected with photosynthetic pigments degradation (mainly chlorophylls) where the pathway of astaxanthin transformation to fatty acid is considered to be the most important process during gall maturation. Nutritive tissue is composed mainly of octadecanoic acid (C18:0) - a main source of food for O. robiniae.

The Floral Signals of the Inconspicuous Orchid Malaxis monophyllos: How to Lure Small Pollinators in an Abundant Environment.

Many orchid species have evolved complex floral signals to ensure pollination efficiency. Here, the authors combined analyses of anatomical flower structure with analyses of the volatile composition and flower-visiting insects' behaviour, as well as characterised features that can attract pollinators of the inconspicuous orchid Malaxis monophyllos. During field observations, the authors found that only small Diptera (e.g., mosquitos, drosophilids, fungus gnats) visit and are interested in the flowers of M. monophyllos, which was reflected in the characterised flower features that combine well with the pollination system, which engages dipterans. Analyses of the M. monophyllos floral scent revealed substantial concentrations of aliphatic compounds, e.g., 1-octen-3-ol and 1-octanol, which condition the mushroom-like scent and a substantial fraction of alkanes, some of which have been previously described as sex mimicry and aggregation pheromones in orchids' deceptive systems. The labellum anatomical structure exhibits a highly diverse cell cuticle surface and pronounced metabolic and secretory activity of the epidermal and subepidermal cells from all parts of the labellum. Moreover, our study provides evidence for the subsequent decoys of M. monophyllos flowers, including visual signals, such as raphides located on the labellum margin and the rewarding ones connected with lipid secretion limited to the area behind the column. Taking an integrative approach to studying M. monophyllos pollination biology, the authors provide new insight into its previously vague pollination strategies and provide evidence for complex floral signal operation in luring potential pollinators. The synergistic effect of M. monophyllos flowers' volatile and visual signals, together with additional rewarding for nectar/fungus/microbe-feeding pollinators, requires further detailed investigation that will be invaluable in explaining the evolution of Diptera-specific pollination systems in orchids.

Irrigation-Induced Changes in Chemical Composition and Quality of Seeds of Yellow Lupine (Lupinus luteus L.).

The quality and amount of yellow lupine yield depend on water availability. Water scarcity negatively affects germination, flowering, and pod formation, and thus introduction of an artificial irrigation system is needed. The aim of this study was to evaluate the influence of irrigation on the quality of yellow lupine seeds. Raining was applied with a semi-solid device with sprinklers during periods of greatest water demand. It was shown that watered plants produced seeds of lesser quality, having smaller size and weight. To find out why seeds of irrigated plants were of poor quality, interdisciplinary research at the cellular level was carried out. DNA cytophotometry evidenced the presence of nuclei with lower polyploidy in the apical zone of mature seeds. This may lead to formation of smaller cells and reduce depositing of storage materials. The electrophoretic and Fourier transform infrared spectroscopic analyses revealed differences in protein and cuticular wax profiles, while scanning electron microscopy and energy dispersive spectroscopy revealed, among various chemical elements, decreased calcium content in one of seed zones (near plumule). Seeds from irrigated plants showed slightly higher germination dynamics but growth rate of seedlings was slightly lower. The studies showed that irrigation of lupine affected seed features and their chemical composition, an ability to germination and seedlings growth.

Irrigation affects characteristics of narrow-leaved lupin (Lupinus angustifolius L.) seeds.

MAIN CONCLUSION: While plant irrigation usually increases yield, irrigation also affects seed characteristics with respect to endoreplication level, chemical composition, number of carbonyl bands, and cuticular wax profiles. Seeds of sweet varieties of the narrow-leaved lupin have good nutritional properties; however, these plants are sensitive to water deficit. Irrigation improves lupin yield, but can affect seed characteristics. The purpose of the study was to evaluate irrigation influence on lupin seed features and their chemical composition. Morphological analyses showed worse quality of seeds from the irrigated plants, with regard to their size and weight. This was confirmed by cytophotometric analyses which revealed a lower DNA content in the nuclei of cells from the apical and basal regions of the irrigated seeds. The lower degree of polyploidy of the nuclei entails lower cell sizes and limited space for storage components. Fourier transform infrared spectroscopic analysis demonstrated that protein and cuticular wax profiles of the irrigated seeds were different from the control. The electrophoretic analyses indicated differences in protein profiles including changes in the proportion of lupin storage proteins. Among the various studied elements, only the nitrogen content decreased in the embryo axis of irrigated plants. Although germination dynamics of the irrigated seeds was higher, the seedlings' development rate was slightly lower than in the control. The hydrogen peroxide level in root meristem cells was higher during germination in the control suggesting its regulatory role in seed metabolism/signaling. Our study indicated that irrigation of lupin plant affected seed features and composition.

Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen.

The phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. We report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation. We applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild-type and ethylene-insensitive hybrid aspen trees (Populus tremula × tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild-type and ethylene-insensitive trees. We demonstrate that ACC and ethylene induce gelatinous layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (Ethylene Response Factors (ERFs), ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1)) and wood formation. G-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

Defense Responses in Aspen with Altered Pectin Methylesterase Activity Reveal the Hormonal Inducers of Tyloses.

Tyloses are ingrowths of parenchyma cells into the lumen of embolized xylem vessels, thereby protecting the remaining xylem from pathogens. They are found in heartwood, sapwood, and in abscission zones and can be induced by various stresses, but their molecular triggers are unknown. Here, we report that down-regulation of PECTIN METHYLESTERASE1 (PtxtPME1) in aspen (Populus tremula × tremuloides) triggers the formation of tyloses and activation of oxidative stress. We tested whether any of the oxidative stress-related hormones could induce tyloses in intact plantlets grown in sterile culture. Jasmonates, including jasmonic acid (JA) and methyl jasmonate, induced the formation of tyloses, whereas treatments with salicylic acid (SA) and 1-aminocyclopropane-1-carboxylic acid (ACC) were ineffective. SA abolished the induction of tyloses by JA, whereas ACC was synergistic with JA. The ability of ACC to stimulate tyloses formation when combined with JA depended on ethylene (ET) signaling, as shown by a decrease in the response in ET-insensitive plants. Measurements of internal ACC and JA concentrations in wild-type and ET-insensitive plants treated simultaneously with these two compounds indicated that ACC and JA regulate each other's concentration in an ET-dependent manner. The findings indicate that jasmonates acting synergistically with ethylene are the key molecular triggers of tyloses.

Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood.

Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.

Reduced expression of the SHORT-ROOT gene increases the rates of growth and development in hybrid poplar and Arabidopsis.

SHORT-ROOT (SHR) is a well characterized regulator of cell division and cell fate determination in the Arabidopsis primary root. However, much less is known about the functions of SHR in the aerial parts of the plant. In this work, we cloned SHR gene from Populus trichocarpa (PtSHR1) as an AtSHR ortholog and down-regulated its expression in hybrid poplar (Populus tremula×P. tremuloides Michx-clone T89) in order to determine its physiological functions in shoot development. Sharing a 90% similarity to AtSHR at amino acid level, PtSHR1 was able to complement the Arabidopsis shr mutant. Down regulation of PtSHR1 led to a strong enhancement of primary (height) and secondary (girth) growth rates in the transgenic poplars. A similar approach in Arabidopsis showed a comparable accelerated growth and development phenotype. Our results suggest that the response to SHR could be dose-dependent and that a partial down-regulation of SHR could lead to enhanced meristem activity and a coordinated acceleration of plant growth in woody species. Therefore, SHR functions in plant growth and development as a regulator of cell division and meristem activity not only in the roots but also in the shoots. Reducing SHR expression in transgenic poplar was shown to lead to significant increases in primary and secondary growth rates. Given the current interest in bioenergy crops, SHR has a broader role as a key regulator of whole plant growth and development and SHR suppression has considerable potential for accelerating biomass accumulation in a variety of species.

Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus.

Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula x tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up- and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.