•Pinus pinaster contains at least four class I and two class II KNOX genes.•First time that functional analyses of class II members are reported in conifers.•Class I genes are expressed in ...meristematic regions and differentiating tissues.•Expression of class II genes is restricted to lateral organs and mature tissues.•Functional differentiation of both subfamilies may be conserved across seed plants.
KNOTTED1-LIKE HOMEOBOX (KNOX) genes are a family of plant-specific homeobox transcription factors with important roles in plant development that have been classified into two subfamilies with differential expression domains and functions. Studies in angiosperms have shown that class I members are related to the maintenance of meristem homeostasis and leaf development, whereas class II members promote differentiation of tissues and organs. However, little is known about its diversification and function in gymnosperms. By combining PCR-based detection and transcriptome data analysis, we identified four class I and two class II KNOX genes in Pinus pinaster. Expression analyses showed that class I members were mainly expressed in meristematic regions and differentiating tissues, with practically no expression in lateral organs, whereas expression of class II members was restricted to lateral organs. Furthermore, overexpression of P. pinaster KNOX genes in Arabidopsis thaliana caused similar phenotypic effects to those described for their angiosperms counterparts. This is the first time to our knowledge that functional analyses of class II members are reported in a conifer species. These results suggest a high conservation of the KNOX gene family throughout seed plants, as the functional differentiation of both subfamilies observed in angiosperms might be partially conserved in gymnosperms.
•Development of a fast and simple method for plant growth regulators analysis.•Avoids the use of tedious and highly time consuming steps.•Feasible to obtain 20 different compounds in only one round ...of extraction.•Capable of quantify in one injection all analytes extracted.•Optimized in complex plant matrices.
Plant growth regulators (PGRs) are very different chemical compounds that play essential roles in plant development and the regulation of physiological processes. They exert their functions by a mechanism called cross-talk (involving either synergistic or antagonistic actions) thus; it is for great interest to study as many PGRs as possible to obtain accurate information about plant status. Much effort has been applied to develop methods capable of analyze large numbers of these compounds but frequently excluding some chemical families or important PGRs within each family. In addition, most of the methods are specially designed for matrices easy to work with. Therefore, we wanted to develop a method which achieved the requirements lacking in the literature and also being fast and reliable. Here we present a simple, fast and robust method for the extraction and quantification of 20 different PGRs using UHPLC–MS/MS optimized in complex matrices.
Conifers are a group of woody plants with an enormous economic and ecological importance. Breeding programs are necessary to select superior varieties for planting, but they have many limitations due ...to the biological characteristics of conifers. Somatic embryogenesis (SE) and de novo organogenesis (DNO) from in vitro cultured tissues are two ways of plant mass propagation that help to overcome this problem. Although both processes are difficult to achieve in conifers, they offer advantages like a great efficiency, the possibilities to cryopreserve the embryogenic lines, and the ability of multiplying adult trees (the main bottleneck in conifer cloning) through DNO. Moreover, SE and DNO represent appropriate experimental systems to study the molecular bases of developmental processes in conifers such as embryogenesis and shoot apical meristem (SAM) establishment. Some of the key genes regulating these processes belong to the
and
homeobox gene families, whose function has been widely described in
. The sequences and roles of these genes in conifers are similar to those found in angiosperms, but some particularities exist, like the presence of
, a gene that putatively participates in the establishment of SAM in somatic embryos and plantlets of
.
Key message
Several members of
WOX
and
KNOX
gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer
Pinus ...pinea
.
Similar to
Arabidopsis thaliana
,
Pinus pinea
shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in
P. pinea
cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in
P. pinea
. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2–6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes
PpWUS
,
PpWOX5
,
PpKN2
,
PpKN3
and
PipiRR1
.
WUS
is functional in pines and has an important role in caulogenesis. Interestingly,
WOX5
also seems to participate in the process, although its specific role has not been determined.
WUSCHEL-RELATED HOMEOBOX (WOX) genes are key players controlling stem cells in plants and can be divided into three clades according to the time of their appearance during plant evolution. Our ...knowledge of stem cell function in vascular plants other than angiosperms is limited, they separated from gymnosperms ca 300 million years ago and their patterning during embryogenesis differs significantly. For this reason, we have used the model gymnosperm Pinus pinaster to identify WOX genes and perform a thorough analysis of their gene expression patterns. Using transcriptomic data from a comprehensive range of tissues and stages of development we have shown three major outcomes: that the P. pinaster genome encodes at least fourteen members of the WOX family spanning all the major clades, that the genome of gymnosperms contains a WOX gene with no homologues in angiosperms representing a transitional stage between intermediate- and WUS-clade proteins, and that we can detect discrete WUS and WOX5 transcripts for the first time in a gymnosperm.
•Pinus pinaster genome encodes at least fourteen members of the WOX family spanning all the major clades.•Discrete WUS and WOX5 transcripts were detected for the first time in a gymnosperm.•Gymnosperm WOXX gene represents a transitional evolutionary stage from intermediate- to WUS-clade proteins.
An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which ...contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.
The molecular cloning and characterization of PipsRR1, a type-A response regulator in Pinus pinaster, is reported here. Type-A response regulators mediate downstream responses to cytokinin and act as ...negative feedback regulators of the signal transduction pathway. Some type-A response regulators in Arabidopsis have been related to de novo meristem formation. However, little information exists in Pinus spp. The PipsRR1 gene contains 5 exons, as do all type-A response regulators in Arabidopsis, and the deduced protein contains a receiver domain with the conserved DDK residues and a short C terminal extension. Expression analysis showed that the PipsRR1 gene is differentially expressed during the first phases of adventitious caulogenesis induced by benzyladenine in P. pinaster cotyledons, suggesting that PipsRR1 plays a role in caulogenesis in conifers. Additionally, a binary vector carrying the PipsRR1 promoter driving GFP:GUS expression was constructed to analyze the promoter activity in P. pinaster somatic embryos. The results of genetic transformation showed GUS activity during somatic embryo mass proliferation and embryo maturation.
Pinus pinaster is one of the most economically important conifers in the world. Somatic embryogenesis is a powerful tool in breeding programmes because it allows the generation of a great number of ...different clonal lines from seeds of superior genotypes. Unfortunately, embryogenic
competence decreases with the age of cultures. Therefore, it is necessary to have a cryopreservation protocol that ensures a continuous supply of juvenile mass while allowing good maturation and conversion rates into vigorously growing plants. In this work we studied the influence of several
cryopreservation parameters, such as cryoprotectant solution and pre-cooling temperature, on embryogenic culture regrowth and embryo maturation. Recovery of rewarmed samples after cryopreservation in a −150°C freezer depended on the cooling temperature reached prior to plunging the
tubes into liquid nitrogen. As a result, we present an optimised cryopreservation protocol that ensures high recovery and embryo maturation rates. The protocol presented is a simple and fast alternative and enabled successful cryopreservation and recovery of 100% of the lines tested. Cryopreserved
lines presented the same maturation rates as non-cryopreserved controls.
Molecular cloning and characterization of a
CLAVATA1
-
LIKE
gene in
Pinus pinaster
and
Pinus pinea
is reported.
CLAVATA1
is a well-characterized gene in
Arabidopsis
integral to the balance between ...primordial differentiation and meristem proliferation. Currently, it is not known if the
Arabidopsis
model of in vitro caulogenesis is applicable to conifers. In this work, we study the putative role of the
CLAVATA1
-
LIKE
gene during caulogenic induction in cotyledons of
P. pinea
and
P. pinaster
after exposure to benzyladenine. Expression analysis showed that the gene was differentially expressed during the first phases of adventitious caulogenesis in cotyledons from both species, suggesting that
CLAVATA1
-
LIKE
may play a role in caulogenesis in conifers. A binary vector carrying
CLAVATA1
-
LIKE
promoter driven GFP:GUS expression was constructed. Results of genetic transformation showed GUS activity during somatic embryogenic mass proliferation and embryo maturation.
Somatic embryogenesis is expected to play a significant role in the future forest tree improvement programmes. The main bottleneck of this technique is still the progression from immature embryogenic ...cultures to mature cotyledonary embryos able to develop properly into well-growing plants. In this work, we present an improved protocol focused on increasing the maturation and conversion rate of Pinus pinaster Ait. embryogenic cultures. Results showed that the optimisation of the nutrient composition in the maturation medium increased the number of mature embryos by 25% (187.8 embryos per gram of fresh mass in average compared to 144.5 embryos in regular medium). It was also shown that 12-month cryostorage did not reduce viability or embryogenic ability of maritime pine cultures. A further increase in the yield of the protocol could be obtained by using benzyladenine in the conversion medium, promoting the bud-break of axillary buds that yielded 5.7 shoots in average per somatic embryo. Rooting of axillary shoots reached 84.3%. This methodology offers an alternative to overcome some problems associated with low somatic embryo production since the plantlet yield could be increased fivefolds.
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