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DRIVING ALONG A COUNTRY LANE to Luc Engelhard’s garden, through lush pastures bounded by rows of pollarded willows, the hustle of the busy main road from Amsterdam to Utrecht behind you is soon forgotten. Luc, a garden designer, and his wife Christine came to this peaceful spot amid the water meadows of Spengen, 15km northwest of central Utrecht, in 1999. The 32-acre plot they bought then consisted of pastures, an orchard and a plantation of 50,000 conifers – the legacy of a nursery once based here. A derelict chicken shed and a few huts were the only buildings. Despite – or perhaps because of – its stark simplicity, Luc liked the feel of this place. He was soon living in a hut on site while he set about landscaping and planting the garden. An old shed became a makeshift office and canteen while he built a house and permanent office.
Creating the garden took all Luc’s experience as a garden designer – 17 years then, 25 now. There were practical considerations: the farm was 1.5m below sea level and its soil made of clay and peat. But Luc decided to make a virtue of this saturated terrain.
“In designing I always bring in elements of the surroundinglandscape,”Lucsays.“Herewaterisoneof the most important elements.” His design includes a series of ponds and waterways. One reaches as far as the foundations of the office, its still surface nearly touching the windows. From the office there is an uninterrupted view over the pond to the garden and the fields beyond. “The wonderful thing with water is that it changes with the weather and the skies above,” says Luc. “The reflec- tions of plants, trees, sculptures and buildings make it all the more fascinating. It is important to experience the seasons intensely in your own garden.”

FURNITURE
Throughout the garden are beautifully designed tables, chairs, loungers, outdoor fireplaces, garden decorations and sculptures. Some are weather-beaten, others brand new – all are for sale, for the garden functions as an open-air showroom for Luc’s own collection and works by leading designers, including Moooi, Dedon, Extremis, Fermob and Domani.
Luc likes combining new and old natural materials in his furniture and buildings as well as in the hard landscaping of his gardens. For example, one of his tableshasastonetopandabrickworkbase,withapair of crude stools made from basalt blocks that were once part of one of the piers in Rotterdam harbour. One of his favourite materials is concrete, which he uses to make floors, table tops, chairs, bowls and vases.

STRIKING FEATURES
Sculptural forms are to be found all over the garden. Emerging from the water at the front of the house is one of the garden’s most striking features, a row of 30 low stone pillars (see page 57) built by Luc from paving stones cut into pieces and stacked in a spiral formation. The pillars seem to move and turn as you view them from different angles, an effect emphasised by changes in the sunlight and the reflections of the water. Such subtle and unexpected effects are deliberate. Luc says: “I like gardens that are not too predictable. There must always be something to discover. A garden must evoke a certain tension, a feel of expectation; a garden must surprise.”
Indeed, a walk through Luc’s garden is full of unex- pected pleasures. Paths lead everywhere, from the orchard and through a former pasture, where grasses are left to grow, to a rustic pergola made of recycled wood. A path of giant stone slabs laid almost haphaz- ardly leads to the waterside, opposite a field of grazing sheep. Another route winds its way to a little bridge; intrepid explorers with a good sense of balance can cross the water via a 40m chestnut boardwalk. On hot summer days Luc’s children like to jump into the water from a tree house in the overhanging boughs of a common alder (Alnus glutinosa) above it.

In the countryside not far from Amsterdam, Dutch designer Luc Engelhard has transformed a once-neglected plot into a beautiful garden full of light, water and space.
Words Thea Seinen, photographs Maayke de Ridder

INSPIRED PLANTING
Luc’s talent for furniture-making is obvious when you visit this garden but so too is his skill as a plantsman, the fruit of a spell at horticultural college and years of trial-and-error in the garden. He says he is always trying new plant combinations. The prairie-style planting in one sunny spot, for example, features Rosa‘Mermaid’,withitscreamy-yellowsingleflowers, scrambling through the upright clumps of purple moor grass (Molinia caerulea ‘Moorhexe’) and airy blue spikes of the relatively low-growing and wind- resistant Delphinium ‘Finsteraarhorn’. Purple Salvia x superba ‘Dear Anja’ provides another vertical accent, while the daisy-like flowers of Echinacea ‘White Swan’ tumble here and there. A shady bed combines false Solomon’s seal (Maianthemum racemosum), the glau- cous leaves of Hosta ‘Krossa Regal’ and the dense, mat-forming grass Luzula sylvatica ‘Bromel’.
Luc enjoys nurturing trees, too. He says he will even adopt trees that are no longer wanted because they have been neglected or badly pruned. “Structure, texture, bark, foliage, seasonal highlights and the way the foliage filters the light are my criteria when selecting trees,” he says. “I like trees thatsay‘thisisthewayIwanttogrow’,andgeton with it. I love willow and hawthorn. I always compare willows with teenagers,” says Luc, who has two children, aged 14 and 12. “They can show such unpredictable behaviour. One moment they are intensely annoying, and the next they are so incredibly nice you truly adore them.
“A willow has the capacity to rejuvenate itself constantly and becomes even more beautiful while aging. Hawthorns are heavily underestimated. In fact they are real fighters that manage to survive and grow under the most difficult circumstances.”

Branches and flower buds were collected from four different sweet cherry cultivars with contrasted flowering dates: ‘Cristobalina’, ‘Garnet’, ‘Regina’ and ‘Fertard’, which display extra-early, early, late and very late flowering dates, respectively. ‘Cristobalina’, ‘Garnet’, ‘Regina’ trees were grown in an orchard located at the Fruit Experimental Unit of INRA in Bourran (South West of France, 44° 19′ 56′′ N, 0° 24′ 47′′ E), under the same agricultural practices. ‘Fertard’ trees were grown in an orchard at the Fruit Experimental Unit of INRA in Toulenne, near Bordeaux (48° 51′ 46′′ N, 2° 17′ 15′′ E). During the first sampling season (2015/2016), ten or eleven dates spanning the entire period from flower bud organogenesis (July 2015) to bud break (March 2016) were chosen for RNA sequencing (Fig. 1a and Additional file 2: Table S1), while bud tissues from ‘Fertard’ were sampled in 2015/2016 (12 dates) and 2017/2018 (7 dates) for validation by RT-qPCR (Additional file 2: Table S1). For each date, flower buds were sampled from different trees, each tree corresponding to a biological replicate. Upon harvesting, buds were flash frozen in liquid nitrogen and stored at − 80 °C prior to performing RNA-seq.

Marker genes were not selected based on modelling fit, nor based on their function.

The seven marker genes were selected based on the following criteria:

Measurements of bud break and estimation of the dormancy release date

Previous studies have proved the key role of a complex array of signaling pathways in the regulation of endodormancy onset and maintenance that subsequently lead to dormancy release, including genes involved in cold response, phytohormone-associated pathways and oxidation-reduction processes. Genes associated with the response to cold, notably, have been shown to be up-regulated during endodormancy such as dehydrins and DREB genes identified in oak, pear and leafy spurge [24, 27, 60]. We observe an enrichment for GO involved in the response to abiotic and biotic responses, as well as an enrichment for targets of many TFs involved in the response to environmental factors. In particular, our results suggest that PavMYB14, which has a peak of expression in November just before the cold period starts, is repressing genes that are subsequently expressed during ecodormancy. This is in agreement with the fact that AtMYB14, the PavMYB14 homolog in Arabidopsis thaliana, is involved in cold stress response regulation [42]. Although these results were not confirmed in Populus [61], two MYB DOMAIN PROTEIN genes (MYB4 and MYB14) were also up-regulated during the induction of dormancy in grapevine [62]. Similarly, we identified an enrichment in genes highly expressed during endodormancy with target motifs of a transcription factor belonging to the CBF/DREB family. These TFs have previously been implicated in cold acclimation and endodormancy in several perennial species [60, 63]. These results are in agreement with the previous observation showing that genes responding to cold are differentially expressed during dormancy in other tree species [24]. Cold acclimation is the ability of plants to adapt to and withstand freezing temperatures and is triggered by decreasing temperatures and photoperiod. Therefore mechanisms associated with cold acclimation are usually observed concomitantly to the early stages of endodormancy. The stability of membranes and a strict control of cellular homeostasis are crucial in the bud survival under cold stress and we observe that genes associated with cell wall organization and nutrient transporters are up-regulated at the beginning of endodormancy, including the CELLULOSE SYNTHASE-LIKE G3 (PavCSLG3) marker gene.

When projected into a PCA 2-components plane, all samples harvested from buds at the same stage cluster together, whatever the cultivar (Fig. 6 and Additional file 1: Figure S5), suggesting that the stage of the bud has more impact on the transcriptional state than time or external conditions. Interestingly, the 100 genes that contributed the most to the PCA dimensions 1 and 2 were very specifically associated with each dimension (Additional file 1: Figure S6, Additional file 2: Table S5). We further investigated which clusters were over-represented in these genes (Additional file 1: Figure S6b) and we found that genes belonging to the clusters 6 and 8, associated with endodormancy, were particularly represented in the best contributors to the dimension 1. In particular, we identified genes involved in oxidation-reduction processes like PavGPX6, and stress-induced genes such as PavLEA14, together with genes potentially involved in leaf and flower development, including GROWTH-REGULATING FACTOR7 (PavGRF7) and PavSEP1 (Table S5). In contrast, genes that best contributed to the dimension 2 strictly belonged to clusters 9 and 10, therefore characterized by high expression during ecodormancy (Additional file 1: Figure S6). These results suggest that bud stages can mostly be separated by two criteria: dormancy depth before dormancy release, defined by genes highly expressed during endodormancy, and the dichotomy defined by the status before/after dormancy release.

In order to predict the bud stage based on the marker genes transcriptomic data, we used TPM values for the marker genes to train and test several models. First, all samples were projected into a 2-dimensional space using PCA, to transform potentially correlated data to an orthogonal space. The new coordinates were used to train and test the models to predict the five bud stage categories. In addition, we tested the model on RT-qPCR data for samples harvested from the ‘Fertard’ cultivar. For the modelling purposes, expression data for the seven marker genes were normalized by the expression corresponding to the October sample. We chose the date of October as the reference because it corresponds to the beginning of dormancy and it was available for all cultivars. For each date, the October-normalized expression values of the seven marker genes were projected in the PCA 2-dimension plan calculated for the RNA-seq data and they were tested against the model trained on ‘Cristobalina’, ‘Garnet’ and ‘Regina’ RNA-seq data.

A list of predicted regulation between transcription factors and target genes is available for peach in PlantTFDB [37]. We collected the list and used it to analyse the overrepresentation of genes targeted by TF, using Hypergeometric available in R, comparing the number of appearances of a gene controlled by one TF in one cluster against the number of appearances on the overall set of DEG. p-values obtained were corrected using a false discovery rate as described above. We only present results obtained for TFs that are themselves DEGs. Predicted gene homology to Arabidopsis thaliana and functions were retrieved from the data files available for Prunus persica (GDR, https://www.rosaceae.org/species/prunus_persica/genome_v2.0.a1).