Tuula Puhakainen

Overexpression of multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis

To elucidate the contribution of dehydrins (DHNs) to freezing stress tolerance in Arabidopsis, transgenic plants overexpressing multiple DHN genes were generated. Chimeric double constructs for expression of RAB18andCOR47(pTP9) or LTI29 and LTI30(pTP10) were made by fusing the coding sequences of the respective DHN genes to the cauliflower mosaic virus 35S promoter. Overexpression of the chimeric genes in Arabidopsis resulted in accumulation of the corresponding dehydrins to levels similar or higher than in cold-acclimated wild-type plants. Transgenic plants exhibited lower LT50 values and improved survival when exposed to freezing stress compared to the control plants. Post-embedding immuno electron microscopy of high-pressure frozen, freeze-substituted samples revealed partial intracellular translocation from cytosol to the vicinity of the membranes of the acidic dehydrin LTI29 during cold acclimation in transgenic plants. This study provides evidence that dehydrins contribute to freezing stress tolerance in plants and suggests that this could be partly due to their protective effect on membranes.

Short-Day Potentiation of Low Temperature-Induced Gene Expression of a C-Repeat-Binding Factor-Controlled Gene during Cold Acclimation in Silver Birch

Development of winter hardiness in trees is a two-stage process involving sequential perception of distinct environmental cues, short-day (SD) photoperiod and low temperature (LT). We have shown that both SD and LT are recognized by leaves of silver birch (Betula pendula cv Roth) leading to increased freezing tolerance, and thus leaves can be used as an experimental model to study the physiological and molecular events taking place during cold acclimation. To obtain a molecular marker for the acclimation process in birch we cloned a gene, designated Bplti36, encoding a 36-kD acidic SK2 type of dehydrin. The gene was responsive to LT, drought, salt, and exogenous abscisic acid. This responsiveness to abiotic stresses and abscisic acid was retained when Bplti36 was introduced to Arabidopsis (Arabidopsis thaliana). The LT induction of the gene appeared to be under the control of the C-repeat-binding factor pathway as suggested by the presence of several C-repeat/dehydration-responsive element/LT-responsive elements in the Bplti36 promoter and its constitutive expression in C-repeat-binding factor overproducing Arabidopsis. In birch SD photoperiod at normal-growth temperature did not result in significant induction of Bplti36. However, preexposure to SD followed by LT treatment resulted in a remarkable increase in Bplti36 transcript accumulation as compared to LT-treated plants grown at long-day photoperiod. This suggests that SD photoperiod potentiates the LT response by conditioning the leaf tissue to be more responsive to the LT stimulus.

Non-Cell-Autonomous Postmortem Lignification of Tracheary Elements in Zinnia elegans

Here, we show that lignification occurs after programmed cell death in xylem tracheary elements (TEs) of Zinnia elegans xylogenic cell cultures. Living, parenchymatic xylem cells surrounding the TEs synthesize and transport lignin monomers and reactive oxygen species to the cell walls of the dead TEs, thereby contributing to TE lignification in a non-cell-autonomous manner. Postmortem lignification of xylem tracheary elements (TEs) has been debated for decades. Here, we provide evidence in Zinnia elegans TE cell cultures, using pharmacological inhibitors and in intact Z. elegans plants using Fourier transform infrared microspectroscopy, that TE lignification occurs postmortem (i.e., after TE programmed cell death). In situ RT-PCR verified expression of the lignin monomer biosynthetic cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase in not only the lignifying TEs but also in the unlignified non-TE cells of Z. elegans TE cell cultures and in living, parenchymatic xylem cells that surround TEs in stems. These cells were also shown to have the capacity to synthesize and transport lignin monomers and reactive oxygen species to the cell walls of dead TEs. Differential gene expression analysis in Z. elegans TE cell cultures and concomitant functional analysis in Arabidopsis thaliana resulted in identification of several genes that were expressed in the non-TE cells and that affected lignin chemistry on the basis of pyrolysis–gas chromatography/mass spectrometry analysis. These data suggest that living, parenchymatic xylem cells contribute to TE lignification in a non-cell-autonomous manner, thus enabling the postmortem lignification of TEs.

Ecotype-dependent control of growth, dormancy and freezing tolerance under seasonal changes in Betula pendula Roth

Abstract. Woody plants in the temperate and boreal zone undergo annual cycle of growth and dormancy under seasonal changes. Growth cessation and dormancy induction in autumn are prerequisites for the development of substantial cold hardiness in winter. During evolution, woody plants have developed different ecotypes that are closely adapted to the local climatic conditions. In this study, we employed distinct photoperiodic ecotypes of silver birch (Betula pendula Roth) to elucidate differences in these adaptive responses under seasonal changes. In all ecotypes, short day photoperiod (SD) initiated growth cessation and dormancy development, and induced cold acclimation. Subsequent low temperature (LT) exposure significantly enhanced freezing tolerance but removed bud dormancy. Our results suggested that dormancy and freezing tolerance might partially overlap under SD, but these two processes were regulated by different mechanisms and pathways under LT. Endogenous abscisic acid (ABA) levels were also altered under seasonal changes; the ABA level was low during the growing season, then increased in autumn, and decreased in winter. Compared with the southern ecotype, the northern ecotype was more responsive to seasonal changes, resulting in earlier growth cessation, cold acclimation and dormancy development in autumn, higher freezing tolerance and faster dormancy release in winter, and earlier bud flush and growth initiation in spring. In addition, although there was no significant ecotypic difference in ABA level during growing season, the rates and degrees of ABA alterations were different between the ecotypes in autumn and winter, and could be related to ecotypic differences in dormancy and freezing tolerance.

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Adaptation of Boreal Field Crop Production to Climate Change

The average annual global temperature increased by 0.76°C during the past century (Intergovernmental Panel on Climate Change (IPCC), 2007) and climate modelling results show an increase in annual temperature in boreal regions of 0.1 – 0.4°C/decade over the 21st century, depending on the scenario and model. Warming will be unevenly distributed, being greater in summer in lower and middle latitudes but greater in winter at higher latitudes, and this differential will increase. Mean annual precipitation is projected to increase in the North and decrease in the South, and winter precipitation will increase in northern and central Europe, continuing the trends established in the 20th century of a 10 – 40% increase in northern Europe and a decrease of up to 20% in southern Europe (IPCC, 2007). The increase in winter precipitation is due to the increased water carrying capacity of the atmosphere resulting from the higher temperature. Global warming will increase the frequency of soil freeze-thaw cycles (FTCs) in cooltemperate and high-latitude regions previously subject to prolonged winter soil frost (Kreyling et al., 2007; Henry, 2008). Warmer winters will result in fewer soil freezing days and in boreal Europe, lowland permafrost is expected to eventually disappear (Harris et al., 2009). The length of the frost-free season has already increased in most midand highlatitude regions of both hemispheres over the values established in the middle of the 20th century. In the Northern Hemisphere, this is mostly manifested as an earlier start to spring, which will arrive progressively earlier in Europe by 2.5 d per decade (Menzel et al., 2006). Increased precipitation in winter, when there is little plant growth, increases the probability of leaching, runoff and erosion from unprotected boreal soils. Climatic warming can paradoxically lead to colder soil temperatures in winter when it reduces the thickness of the insulating snow cover (Henry, 2008 and references therein) leading to root injury (Kreyling, 2010). Increased soil freezing when snow was removed led to root injury, increased leaching of C, N and P, and decreased soil microarthropod abundance (Groffman et al., 2001; Weih & Karlsson, 2002; Henry, 2008), but it is unclear what impacts FTCs and lower soil temperature will have on soil biological and physical processes. Observed nitrate losses to the groundwater after deep soil frost events are attributable more to reduced root uptake due to root injury than to increased N net mineralization (Matzner & Borken, 2008) (Figure 1). The intensity and frequency of summer heat waves is likely to increase (IPCC, 2007). Between 1977 and 2000, these trends were more extreme in central and north-eastern Europe and in mountainous regions than in the Mediterranean region. Temperatures are increasing

Engineering Trehalose Biosynthesis Improves Stress Tolerance in Arabidopsis

Environmental stresses caused by drought and extremes of temperature are main factors limiting plant growth, productivity and distribution and up to 80 % of the total crop losses are caused by such climatic factors (Boyer, 1982). Consequently, increase in plant stress tolerance could have a major impact on agricultural productivity. Genetic engineering has recently been shown to provide new approaches for plant breeding and considerable efforts has been made to design strategies for genetic engineering of stress tolerance (Thomashow, 1999; Nuotio et al., 2001). Unfortunately, developmental, structural and physiological adaptations to stresses are often based on complex mechanisms involving a number of different genes (McCue and Hanson, 1990) and therefore not amenable to genetic engineering. However, some of the responses to abiotic stress appear to be based on relatively simple metabolic traits governed by a limited number of genes.