Patricio Ramos

Transcriptional analysis of differentially expressed genes in response to stem inclination in young seedlings of pine.

The gravitropic response in trees is a widely studied phenomenon, however understanding of the molecular mechanism involved remains unclear. The purpose of this work was to identify differentially expressed genes in response to inclination using a comparative approach for two conifer species. Young seedlings were subjected to inclination and samples were collected at four different times points. First, suppression subtractive hybridisation (SSH) was used to identify differentially regulated genes in radiata pine (Pinus radiata D. Don). cDNA libraries were constructed from the upper and lower part of inclined stems in a time course experiment, ranging from 2.5 h to 1 month. From a total of 3092 sequences obtained, 2203 elements were assembled, displaying homology to a public database. A total of 942 unigene elements were identified using bioinformatic tools after redundancy analysis. Of these, 614 corresponded to known function genes and 328 to unknown function genes, including hypothetical proteins. Comparative analysis between radiata pine and maritime pine (Pinus pinaster Ait.) was performed to validate the differential expression of relevant candidate genes using qPCR. Selected genes were involved in several functional categories: hormone regulation, phenylpropanoid pathway and signal transduction. This comparative approach for the two conifer species helped determine the molecular gene pattern generated by inclination, providing a set of Pinus gene signatures that may be involved in the gravitropic stress response. These genes may also represent relevant candidate genes involved in the gravitropic response and potentially in wood formation.

A differential distribution of auxin and flavonols in radiata pine stem seedlings exposed to inclination

Stem reorientation in response to inclination in trees has commanded the attention of the research community widely, but the molecular mechanisms of the response are still unknown. The most accepted theory is auxin redistribution in affected tissues. Flavonols have been reported as potent inhibitors of polar auxin transport; however, no reports have shown differential modulation by flavonols within the trunk. Here, transcriptional, microscopic, and chemical approaches were used to analyze the contents of flavonoids in 45° inclined radiata pine seedlings. A time course was established and samples obtained by cutting stems into apical, medial, and basal segments or by longitudinal dissection into upper and lower halves to determine polar distribution of flavonoids. The expression analysis of PrCHS, PrF3H, and PrFLS indicated induction in response to inclination; higher transcript levels were recorded at the basal zone and in the upper half of the stem. A reduction in abundance of PrARP transcripts in addition to immunospecific auxin detection at the lower half of inclined stem was found. Concomitantly, quercetin and kaempferol accumulated in the upper stem half. Our results suggest that stem reorientation is a response to a concerted and differential mechanism that modulates imbalance of auxin distribution in one side of the stem.

MYB Transcription Factors and a Putative Flavonoid Transporter ABCC-Like are Differentially Expressed in Radiata Pine Seedlings Exposed to Inclination

The response to inclination in plants is a well-studied biological process where hormones have been implicated in the modulation of vertical growth restoration. Studies on the involvement of auxins in differential growth of plant stems have resulted in the hypothesis that differential growth in stem tissue is due to unequal auxin re-distribution. It is known that flavonoids affect auxin distribution, but how the intracellular flavonoid concentration is controlled during the gravitropic response is unknown, especially in tree species. Due to the relationship between auxin, flavonoids, and transport during the response to inclination, genes coding for the control of flavonoid levels were identified and transcriptionally characterized in the transcriptome of pine seedlings exposed to inclination. Four MYB transcripts and one transcript encoding a transporter called PrMRP1 were identified and confirmed using qPCR. PrMRP1 has a high identity with specific transporters of flavonoids; its subcellular localization by fusion with green fluorescent protein and transient expression in tobacco was analyzed, confirming its location in the vacuolar membrane. These results suggest the involvement of MYBs and this transporter in regulating the distribution of flavonoids on the upper side of the stem at early stages of inclination.

PrMATE1 Is Differentially Expressed in Radiata Pine Exposed to Inclination and the Deduced Protein Displays High Affinity to Proanthocyanidin Substrates by a Computational Approach

The response to inclination in plants is an attractive and extensively studied biological process. The most commonly held theory proposes a differential growth in stem tissue due to unequal auxin redistribution. Further evidence proposed that flavonoids act as molecular regulators of auxin distribution or flux. It is well known that flavonoids affect auxin distribution, but how intracellular concentration is controlled during the gravitropic response in woody species is still unknown. The MATE family has been widely studied, however the molecular basis of flavonoids transport is still poorly understood. Here, we identified and characterized a full-length cDNA from radiate pine encoding a putative MATE protein. Transcript abundance analysis showed that PrMATE1 is expressed in a spatial and temporal manner in inclined stems. Additionally, PrMATE1 fused to GFP is mainly localized in the vacuolar membrane. A 3D protein model showed 12 transmembrane helices and an open cavity. The protein–ligand interaction was evaluated; favourable binding affinity energies were obtained and suggested epicatechin 3′-O-glucoside as the best putative ligand. In silico mutagenesis analysis was used to identify five residues as important to protein–ligand interaction. The data provide a dynamic view of interaction between PrMATE1 and their putative ligands at the molecular scale.

Computational study of FaEXPA1, a strawberry alpha expansin protein, through molecular modeling and molecular dynamics simulation studies

Changes in the cellulose-hemicellulose fraction take place during ripening of strawberry fruit and are associated with the activity of a set of proteins and hydrolytic enzymes. Expansins are proteins located in the cell wall with no catalytic activity. In this context, FaEXPA1 was previously reported to have a high accumulation rate during fruit ripening in three different strawberry cultivars. In order to understand at the molecular level the expansin mechanism mode, a 3D model of FaEXPA1 protein was built by comparative modeling. FaEXPA1 protein model displayed two domains, a cellulose-binding domain with a β-sandwich structure, and a second domain that included a HFD motif with a similar structure to the catalytic core of endoglucanase V from Humicola insolens. Additionally, in the center of the structure, an open groove was formed. Finally, using a cellulose polymer as a ligand, the protein-ligand interaction was evaluated by molecular dynamic (MD) simulation. Two MD simulations showed that FaEXPA1 can interact with cellulose via the flat aromatic surface of its binding domain D2, composed mainly of residues Trp99 and Trp225. In addition, FaEXPA1 formed a high number of hydrogen bonds with the glycan chain and the Asn81, Phe114 and Asn211 residues.

Gravitropic response in radiata pine seedlings. Searching molecular keys

Coniferous trees develop compression wood in response to gravitropic stimuli. In nature this response can be generated by growth in slope, exposure to snow or high winds [1]. However, genes and molecular mechanism involved in this phenomenon are still unknown. We studied gene expression in response to gravitropic stimulation induced by 45° inclination in Pinus radiata D. Don one year old seedlings. To characterize the gravitropic response, whole seedlings were inclined and transversal cuts were performed in order to identify morphological wood characteristics. Xylem cells were visualized by optical microscopy in a time course experiment (fig. ​(fig.1).1). On the other hand, a transcriptomic approach was assayed generating libraries based on the Suppressive Subtractive Hybridization (SSH) strategy [2]. This technique allows the isolation of genes differentially expressed between two samples. The libraries were generated from total RNA extracted at 2.5, 10, 24 hours and 30 days from the inferior and superior stem half. The sequences obtained were assembled, analyzed and ontology classified by biological process, molecular function and cell components. The information give clue about the molecular mechanism related to this phenomenon. To validate the differential gene expression by qPCR analyses, housekeeping genes were evaluated in order to have normalization genes for the gravitropic stress response. We could identify a large number of genes activation involved in different initial process, previous to the anatomical hallmarks of compression wood formation. Figure 1 Anatomical modification on lignin deposition and xylem cell shape on inclined seedlings. One-year old Pinus radiata seedlings were inclined during 15 days (d) and transversally cut at 5d, 8d and 15d. Stem slices were stained using a solution of phloroglucinol (2A). From these preparations, wall thickness (2B) and cell diameter (2C) of xylem cells were measured.