Differential expression of poplar <Emphasis Type="Italic">sucrose nonfermenting1-related protein kinase 2</Emphasis> genes in response to abiotic stress and abscisic acid

Knowledge on the responses of woody plants to abiotic stress can inform strategies to breed improved tree varieties and to manage tree species for environmental conservation and the production of lignocellulosic biomass. In this study, we examined the expression patterns of poplar (Populus trichocarpa) genes encoding members of the sucrose nonfermenting1-related protein kinase 2 (SnRK2) family, which are core components of the abiotic stress response. The P. trichocarpa genome contains twelve SnRK2 genes (PtSnRK2.1- PtSnRK2.12) that can be divided into three subclasses (I–III) based on the structures of their encoded kinase domains. We found that PtSnRK2s are differentially expressed in various organs. In MS medium-grown plants, all of the PtSnRK2 genes were significantly upregulated in response to abscisic acid (ABA) treatment, whereas osmotic and salt stress treatments induced only some (four and seven, respectively) of the PtSnRK2 genes. By contrast, soil-grown plants showed increased expression of most PtSnRK2 genes under drought and salt treatments, but not under ABA treatment. In soil-grown plants, drought stress induced SnRK2 subclass II genes in all tested organs (leaves, stems, and roots), whereas subclass III genes tended to be upregulated in leaves only. These results suggest that the PtSnRK2 genes are involved in abiotic stress responses, are at least partially activated by ABA, and show organ-specific responses.     

The role of expansin genes <Emphasis Type="Italic">PtrEXPA3</Emphasis> and <Emphasis Type="Italic">PnEXPA3</Emphasis> in the regulation of leaf growth in poplar

The genes of α-expansins of woody plants are of great interest for genetic engineering, since they can potentially be used to improve the tree growth parameters. In the flora of Russia, model woody plants for plant biotechnology are aspen (Populus tremula L.) and black poplar (Populus nigra L.). The objective of this study was to determine the role of α-expansin-encoding genes, aspen PtrEXPA3 and black poplar PnEXPA3, in the regulation and maintenance of woody plant growth. To achieve this goal, the PtrEXPA3 expression level were determined upon exogenous phytohormone treatment, the action of stress factors, and constitutive expression of the PnARGOS-LIKE gene. In addition, transgenic aspen plants with constitutive expression of the black poplar PnEXPA3 gene were generated, and their morphological analysis was carried out. The highest PtrEXPA3 mRNA level was detected in young intensely growing aspen leaves, and furthermore, expression of the gene was induced by exogenous cytokinins and auxins. In response to NaCl and constitutive expression of the PnARGOS-LIKE gene, the PtrEXPA3 mRNA level decreased. Transgenic aspen plants with constitutive PnEXPA3 expression were characterized by the decreased size of leaves, petioles, and internodes, as well as the increased size of leaf epidermal cells, while the stem size remained unchanged. Taken together, the data obtained enable the suggestion that the PtrEXPA3 and PnEXPA3 genes encode cytokinin- and auxin-regulated, leaf-specific expansins that are involved in the cell expansion.     

The poplar ARGOS-LIKE gene promotes leaf initiation and cell expansion,and controls organ size

We identified a Populus nigra auxin-regulated gene involved in organ size (PnARGOS)-LIKE, encoding one organ size related protein in black poplar. It is homologous to AtARGOS and AtARGOS-LIKE genes of Arabidopsis thaliana. ABRE-like, G-box, GATA and I-box motifs were discovered in the promoter region of the poplar ARGOS-LIKE gene. In wild type aspen (Populus tremula) plants, an ortholog of the PnARGOS-LIKE gene (PtrARGOS-LIKE) was noticeably expressed in actively dividing and expanding young leaves and calli, whereas its mRNA content increased in response to exogenous 6-benzylaminopurine, 1-naphthaleneacetic acid, and 24-epibrassinolide. Expression of the PtrARGOS-LIKE gene was reduced under a salinity treatment. In addition, we generated transgenic tobacco and aspen plants with an up-regulated expression of the PnARGOS-LIKE gene. A constitutive expression of the gene contributed to an increase in size of stems and leaves of the transgenic tobacco plants. In the transgenic aspen, a constitutive expression of the PnARGOS-LIKE gene promoted an increase in the frequency of leaf initiations and in leaf length and area. The size of transgenic tobacco and aspen leaves increased due to the enlargement of individual cells. The results show the significance of the PnARGOS-LIKE gene for control of leaf initiation and organ growth by cell expansion in poplar.     

Comparative genome-wide analysis of repetitive DNA in the genus <Emphasis Type="Italic">Populus</Emphasis> L.

Genome skimming was performed, using Illumina sequence reads, in order to obtain a detailed comparative picture of the repetitive component of the genome of Populus species. Read sets of seven Populus and two Salix species (as outgroups) were subjected to clustering using RepeatExplorer (Novák et al. BMC Bioinformatics 11:378 2010). The repetitive portion of the genome ranged from 33.8 in Populus nigra to 46.5% in Populus tremuloides. The large majority of repetitive sequences were long terminal repeat-retrotransposons. Gypsy elements were over-represented compared to Copia ones, with a mean ratio Gypsy to Copia of 6.7:1. Satellite DNAs showed a mean genome proportion of 2.2%. DNA transposons and ribosomal DNA showed genome proportions of 1.8 and 1.9%, respectively. The other repeat types accounted for less of 1% each. Long terminal repeat-retrotransposons were further characterized, identifying the lineage to which they belong and studying the proliferation times of each lineage in the different species. The most abundant lineage was Athila, which showed large differences among species. Concerning Copia lineages, similar transpositional profiles were observed among all the analysed species; by contrast, differences in transpositional peaks of Gypsy lineages were found. The genome proportions of repeats were compared in the seven species, and a phylogenetic tree was built, showing species separation according to the botanical section to which the species belongs, although significant differences could be found within sections, possibly related to the different geographical origin of the species. Overall, the data indicate that the repetitive component of the genome in the poplar genus is still rapidly evolving.     

Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, <Emphasis Type="Italic">AtSRK2C</Emphasis> and <Emphasis Type="Italic">AtGolS2</Emphasis>

Recent environmental issues have increased the demand for woody biomass as a renewable resource for industry and energy. For a stable supply of woody biomass, it is critical to decrease the effects of abiotic stresses, such as drought and salinity, which hinder plant growth. For the goal to develop practical stress-tolerant trees, we generated transgenic poplar plants (P. tremula × tremuloides), in which a key Arabidopsis regulatory factor involved in stress responses, SNF1-related protein kinase 2C (AtSRK2C), or galactinol synthase 2 (AtGolS2), was overexpressed. Both types of transgenic poplar plants displayed higher tolerance to abiotic stresses, in comparison with nontransgenic plants, indicating that AtSRK2C and AtGolS2 can function in the abiotic stress response pathway of poplar. We also examined the expression profiles of ten poplar genes putatively homologous to well-known Arabidopsis stress response genes and found that several of the poplar genes showed different responses to abiotic stress from their Arabidopsis counterparts. Whereas the overexpression of AtSRK2C in transgenic Arabidopsis plants was reported to upregulate the expression of endogenous genes, the overexpression of AtSRK2C or AtGolS2 in transgenic poplar did not. Taken together, our findings suggest that the details of the underlying molecular mechanisms of the abiotic stress response may differ, but that the key regulatory factors in Arabidopsis and poplar have common features and are effective molecular targets for further breeding to enhance abiotic stress tolerance in poplar.     

Genome sequencing and analysis of <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> strain 34-9, a biocontrol agent against postharvest pathogens in citrus

Kloeckera apiculata, as the anamorphic state of Hanseniaspora uvarum from the Ascomycota phylum, plays an important role in the inhibition of fungal diseases in plants and spontaneous wine fermentation. This study was performed to sequence and analyze the whole genome of K. apiculata strain 34-9; This analysis provides further genomic features and assists functional research. The complete genome was determined using an Illumina HiSeq 2000 system applying paired-end and mate-pair methods to construct four reads libraries. The data assembly of all the reads resulted in a total genome size of 8.1 Mb, including 106 contigs, which were assembled into 41 scaffolds with a 31.95 % G+C content and a 234X sequence coverage. The performance of the gene prediction and functional annotation revealed that 2724 of 3786 protein-coding genes matched the KOG database, and 1127 genes were classified into GO categories. Further genome features analyses found 1066 microsatellite sites, 71 tRNAs, 3 rRNAs and 3 microRNAs in the genomic DNA. A prediction of the metabolic pathways identified potentially crucial genes for explaining the phenylalanine pathway involved in biocontrol. Comparisons with the typical yeasts Lachancea thermotolerans, Kluyveromyces lactis and Saccharomyces cerevisiae revealed the particularity and difference of K. apiculata strain 34-9. The genome alignments among Hanseniaspora vineae T02/19AF, K. apiculata DSM 2768 and 34-9 indicated numerous homologous regions distributed over the genomes between strain DSM2768 and 34-9. A SSR analysis identified that mono- and tri- nucleotide repeat types were more abundant in all six types, likely affecting the evolution of K. apiculata.     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.