SHORT-ROOT (SHR) is a GRAS transcription factor first characterized for its role in the specification of the stem cell niche and radial patterning in Arabidopsis thaliana (At) roots. Three SHR-like genes have been identified in Populus trichocarpa (Pt). PtSHR1 shares high similarity with AtSHR over the entire length of the coding sequence. The two other Populus SHR-like genes, PtSHR2A and PtSHR2B, are shorter in their 5' ends when compared with AtSHR. Unlike PtSHR1, that is expressed throughout the cambial zone of greenhouse-grown Populus trees, PtSHR2Bprom:uidA expression was detected in the phellogen. Additionally, PtSHR1 and PtSHR2B expression patterns markedly differ in the shoot apex and roots of in vitro plants. Transgenic hybrid aspen expressing PtSHR2B under the 35S constitutive promoter showed overall reduced tree growth while the proportion of bark increased relative to the wood. Reverse transcription–quantitative PCR (RT–qPCR) revealed increased transcript levels of cytokinin metabolism and response-related genes in the transgenic plants consistent with an increase of total cytokinin levels. This was confirmed by cytokinin quantification by LC-MS/MS. Our results indicate that PtSHR2B appears to function in the phellogen and therefore in the regulation of phellem and periderm formation, possibly acting through modulation of cytokinin homeostasis. Furthermore, this work points to a functional diversification of SHR after the divergence of the Populus and Arabidopsis lineages. This finding may contribute to selection and breeding strategies of cork oak in which, unlike Populus, the phellogen is active throughout the entire tree lifespan, being at the basis of a highly profitable cork industry.

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 μM abscisic acid (ABA), 9 g l⁻¹ gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.

Regulation of seed development by small non-coding RNAs (sRNAs) is an important mechanism controlling a crucial phase of the life cycle of seed plants. In this work, sRNAs from seed tissues (zygotic embryos and megagametophytes) and from somatic embryos of Pinus pinaster were analysed to identify putative regulators of seed/embryo development in conifers. In total, sixteen sRNA libraries covering several developmental stages were sequenced. We show that embryos and megagametophytes express a large population of 21-nt sRNAs and that substantial amounts of 24-nt sRNAs were also detected, especially in somatic embryos. A total of 215 conserved miRNAs, one third of which are conifer-specific, and 212 high-confidence novel miRNAs were annotated. MIR159, MIR171 and MIR394 families were found in embryos, but were greatly reduced in megagametophytes. Other families, like MIR397 and MIR408, predominated in somatic embryos and megagametophytes, suggesting their expression in somatic embryos is associated with in vitro conditions. Analysis of the predicted miRNA targets suggests that miRNA functions are relevant in several processes including transporter activity at the cotyledon-forming stage, and sulfur metabolism across several developmental stages. An important resource for studying conifer embryogenesis is made available here, which may also provide insightful clues for improving clonal propagation via somatic embryogenesis.

In plants, secondary growth results in radial expansion of stems and roots, generating large amounts of biomass in the form of wood. Using genome-wide association studies (GWAS)-guided reverse genetics in Arabidopsis thaliana, we discovered SOBIR1/EVR, previously known to control plant immunoresponses and abscission, as a regulator of secondary growth. We present anatomical, genetic, and molecular evidence indicating that SOBIR1/EVR prevents the precocious differentiation of xylem fiber, a key cell type for wood development. SOBIR1/EVR acts through a mechanism that involves BREVIPEDICELLUS (BP) and ERECTA (ER), 2 proteins previously known to regulate xylem fiber development. We demonstrate that BP binds SOBIR1/EVR promoter and that SOBIR1/EVR expression is enhanced in bp mutants, suggesting a direct, negative regulation of BP over SOBIR1/EVR expression. We show that SOBIR1/EVR physically interacts with ER and that defects caused by the sobir1/evr mutation are aggravated by mutating ER, indicating that SOBIR1/EVR and ERECTA act together in the control of the precocious formation of xylem fiber development. Significance Secondary growth (thickening of stems and roots) provides the mechanical support that plants need to grow and sustain their structures, and results in wood formation. Although wood accounts for the largest proportion of terrestrial biomass, the identification through classical genetics of key elements that control secondary growth is hampered by the challenge of phenotyping traits associated with inner tissues. We have taken advantage of natural genetic variation to identify SOBIR1/EVR, a gene previously known for its involvement in plant immunity and abscission, as a regulator of the process, and demonstrate that it prevents precocious formation of xylem fiber cells through a mechanism that involves the activity of previously discovered master regulators of xylem fiber development.