Effects of drought stress memory on the accumulation of stress-protective compounds in naturally grown pine and spruce.

Forest trees are subjected to multiple stressors during their long lifetime and therefore require effective and finely regulated stress-protective systems. Stressors can induce protective systems either directly or with the involvement of stress memory mechanisms. Stress memory has only begun to be uncovered in model plants and is unexplored in coniferous species. Therefore, we studied the possible role of stress memory in the regulation of the accumulation of stress-protective compounds (heat shock proteins, dehydrins, proline) in the needles of naturally grown Scots pine and Norway spruce trees subjected to the subsequent action of long-term (multiyear) and short-term (seasonal) water shortages. Although the water deficit was relatively mild, it significantly influenced the pattern of expression of stress memory-related heat shock factor (HSF) and SWI/SNF genes, indicating the formation of stress memory in both species. In spruce, dehydrin accumulation was increased by water shortage in a manner compatible with Type II stress memory. The accumulation of HSP40 in spruce needles was positively influenced by long-term water shortage, but this increase was unlikely to be of biological importance due to the concomitant decrease in HSP70, HSP90 and HSP101 accumulation. Finally, proline accumulation was negatively influenced by short-term water deficit in spruce. In pine, no one protective compound accumulated in response to water stress. Taken together, the results indicate that the accumulation of stress-protective compounds was generally independent of stress memory effects both in pine and in spruce.

Manganese Deficiency Suppresses Growth and Photosynthetic Processes but Causes an Increase in the Expression of Photosynthetic Genes in Scots Pine Seedlings.

Manganese deficiency is a serious plant nutritional disorder, resulting in the loss of crop productivity in many parts of the world. Despite the progress made in the study of angiosperms, the demand for Mn in gymnosperms and the physiological responses to Mn deficiency remain unexplored. We studied the influence of Mn deficiency for 24 weeks on Pinus sylvestris L. seedling growth, ion homeostasis, pigment contents, lipid peroxidation, chlorophyll fluorescence indices and the transcript levels of photosynthetic genes and genes involved in chlorophyll biosynthesis. It was shown that Mn-deficient plants demonstrated suppressed growth when the Mn content in the needles decreased below 0.34 µmol/g DW. The contents of photosynthetic pigments decreased when the Mn content in the needles reached 0.10 µmol/g DW. Mn deficiency per se did not lead to a decrease in the nutrient content in the organs of seedlings. Photoinhibition of PSII was observed in Mn-deficient plants, although this was not accompanied by the development of oxidative stress. Mn-deficient plants had an increased transcript abundance of genes (psbO, psbP, psbQ, psbA and psbC), encoding proteins directly associated with the Mn cluster also as other proteins involved in photosynthesis, whose activities do not depend on Mn directly. Furthermore, the transcript levels of the genes encoding the large subunit of Rubisco, light-dependent NADPH-protochlorophyllide oxidoreductase and subunits of light-independent protochlorophyllide reductase were also increased in Mn-deficient plants.

Cytokinin Perception in Ancient Plants beyond Angiospermae.

Cytokinins (CKs) control many plant developmental processes and responses to environmental cues. Although the CK signaling is well understood, we are only beginning to decipher its evolution. Here, we investigated the CK perception apparatus in early-divergent plant species such as bryophyte Physcomitrium patens, lycophyte Selaginella moellendorffii, and gymnosperm Picea abies. Of the eight CHASE-domain containing histidine kinases (CHKs) examined, two CHKs, PpCHK3 and PpCHK4, did not bind CKs. All other CHK receptors showed high-affinity CK binding (KD of nM range), with a strong preference for isopentenyladenine over other CK nucleobases in the moss and for trans-zeatin over cis-zeatin in the gymnosperm. The pH dependences of CK binding for these six CHKs showed a wide range, which may indicate different subcellular localization of these receptors at either the plasma- or endoplasmic reticulum membrane. Thus, the properties of the whole CK perception apparatuses in early-divergent lineages were demonstrated. Data show that during land plant evolution there was a diversification of the ligand specificity of various CHKs, in particular, the rise in preference for trans-zeatin over cis-zeatin, which indicates a steadily increasing specialization of receptors to various CKs. Finally, this distinct preference of individual receptors to different CK versions culminated in vascular plants, especially angiosperms.

Impact of high irradiance and UV-B on the photosynthetic activity, pro-/antioxidant balance and expression of light-activated genes in Arabidopsis thaliana hy4 mutants grown under blue light.

The impacts of high-intensity light (HIL) (4 h) and UV-B radiation (1 h) on the photosynthetic activity, content of photosynthetic and UV-absorbing pigments (UAPs), activity of antioxidant enzymes (ascorbate peroxidase (APX) and guaiacol-dependent peroxidase (GPX)), content of thiobarbituric acid reactive substances (TBARs), expression of some light-regulated genes in 25-day-old wild type (WT) and the cryptochrome 1 (Cry1) hy4 mutant of A. thaliana Col-0 plants grown under blue light (BL) were studied. HIL and UV-B treatments led to decreases in the photosynthetic rate (Pn), photochemical activity of PSII (FV/FM) and PSII performance index (PIABS) of WT and mutant plants grown under high-intensity BL (HBL) and moderate intensity BL (MBL). However, in HBL plants, the decrease in the photosynthetic activity in hy4 plants was significantly greater than that in WT plants. In addition, hy4 HBL plants demonstrated lowered UAP and carotenoid contents as well as lower activity of APX and GPX enzymes. The difference in the decline in the photosynthetic activity of WT and hy4 plants grown at MBL in response to HIL was nonsignificant, while that in response to UV-B was small. We assume that the deficiency in cryptochrome 1 under HIL irradiation disrupts the interaction between HY5 and HFR1 transcription factors and photoreceptors, which affects the transcription of light-induced genes, such as CAB1, PSY and PAL1 linked to carotenoid and flavonoid biosynthesis. It was concluded that PA stress resistance in WT and hy4 plants depends on the light intensity and reduced stress resistance of hy4 at HBL, is likely linked to low UAP and carotenoid contents as well as lowered APX and GPX enzyme activities in hy4 mutants.

Effect of high-intensity light and UV-B on photosynthetic activity and the expression of certain light-responsive genes in A. thaliana phyA and phyB mutants.

The effects of high-intensity light (HIL, 4 and 24 h) and UV-B (1 h) on the net photosynthesis rate, activity of photosystem II (PSII), content of photosynthetic pigments, anthocyanin and UV-absorbing pigments as well as the expression of certain light-responsive genes (HY5,CAB1) chalcone synthase (CHS) and main antioxidants enzyme genes (APX1, GPX and GR) in the leaves of phyB and phyA mutant A. thaliana plants were studied. Both UV-B and 4 and 24 h HIL decreased the PSII maximum quantum yield (Fv/Fm), PSII performance index (PIABS), photosynthesis and respiration rates in plants. Moreover, all stress treatments increased the dissipation of the absorbed energy (DI0/RC) as well as the flux of absorbed energy per RC (ABS/RC). The maximal changes in photosynthesis and chlorophyll fluorescence parameters were observed in the phyB mutant. The WT and the phyA mutant showed similar responses. In addition, the phyB mutant exhibited decreases in the expression of genes encoding enzyme CHS, the transcription factor HY5 and the antioxidant enzymes APX1 and GPX. One of the possible mechanisms protecting the photosynthetic apparatus from light excess or UV radiation is the elevated content of various pigments that can act as antioxidants or optical filters. We assume that the greater decrease in photosynthetic activity in the phyB mutant under HIL and UV-B conditions was due to the decreased content of carotenoids and UV-absorbing pigments in this mutant.

Quantitative analysis of differential dehydrin regulation in pine and spruce seedlings under water deficit.

Dehydrins are well-known components of plant responses to different stresses that cause dehydration, including drought, freezing, salinity, etc. In conifers, the dehydrin gene family is very large, implying that the members of this family have important physiological functions in conifer stress tolerance. However, dehydrin gene expression displays a wide range of responses to stress, from thousand-fold increased expression to decreased expression, and it is generally unknown how regulatory systems are connected at the mRNA and protein levels. Therefore, we studied these aspects of dehydrin regulation in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) H. Karst) seedlings under polyethylene glycol 6000-induced osmotic stress ranging from relatively low (culture medium water potential of -0.15 MPa) to very high (-1.0 MPa) intensities. In pine, the major dehydrin protein was Dhn1 in both the roots and needles, and in spruce, two isoforms of the Dhn4 protein were the major dehydrins; both of these proteins are AESK-type dehydrins. The genes encoding these major proteins were highly expressed even under control conditions; surprisingly, we also observed several highly expressed dehydrin genes that were not abundantly translated. Under osmotic stress, the most prominent expression changes were observed for the dehydrin genes with low basal expression levels, whereas highly expressed genes generally demonstrated rather modest changes in expression. We report proposed constitutive physiological functions of the AESK-type dehydrins in Pinaceae plants.

Deficiencies in phytochromes A and B and cryptochrome 1 affect the resistance of the photosynthetic apparatus to high-intensity light in Solanum lycopersicum.

The effects of high-intensity light (HIL) on the activity of photosystem II (PSII) and photosynthesis in wild-type (WT) and single (phyB2, phyB1, phyA and cry1), double (phyB1B2, phyAB2 and phyAB1) and triple (phyAB1B2 and cry1phyAB1) mutants of Solanum lycopersicum were studied. In addition, changes in the activity of the antioxidant enzymes ascorbate peroxidase, glutathione reductase and guaiacol peroxidase as well as the photosynthetic pigment and anthocyanin contents in the leaves of phyB2 and cry1phAB1 mutants under HIL were examined. When plants were irradiated with HIL (2 h), the PSII resistance of the cry1phyAB1 mutant was the lowest, while the resistance of WT and single mutants excluding cry1 was the highest. The effect of HIL on PSII activity in all double mutants and the phyAB1B2 mutant was intermediate between the effects on the WT and the cry1phyAB1 mutant. The intensity of oxidative processes in the cry1phyAB1 mutant was higher than that in WT and phyB2, but in cry1phyAB1, the activity of antioxidant enzymes and the anthocyanin content were lower. The low resistance of the cry1phyAB1 mutant to HIL may be due to the low antioxidant activity of key enzymes and the reduced pigment content, which are consistent with the reduced expression of CHS and sAPX genes in the cry1phyAB1 mutant.

Profiles of endogenous phytohormones and expression of some hormone-related genes in Scots pine and Norway spruce seedlings under water deficit.

Different plant hormones are involved in plant adaptation to water deficit. In comparison to angiosperms, little is known about the impact of drought on the pool of phytohormones in gymnosperms. Therefore, we studied the effect of polyethylene glycol-induced water deficit on the changes in content of different phytohormones in Scots pine and Norway spruce seedlings, which are known for their different strategies of adaptation to water deficit. The following hormone classes were analysed: cytokinins, auxins, jasmonates, salicylic and benzoic acids, and 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor). No consistent reaction to water stress was observed for the content of well-known stress-related hormones - salicylic acid and jasmonates. In contrast, drought induced a dose-dependent accumulation of cytokinins in pine needles, with less profound changes in spruce needles. The most prominent changes were observed for 1-aminocyclopropane-1-carboxylic acid content, which increased several-fold in spruce roots and pine needles under water deficit. Water-deficit-induced changes in the contents of cytokinins and 1-aminocyclopropane-1-carboxylic acid were accompanied by the differential regulation of genes involved in the metabolism of these hormones. Possible links between changes in hormone pools and the adaptation of seedlings to water deficit are discussed.

Comparative photosynthetic responses of Norway spruce and Scots pine seedlings to prolonged water deficiency.

Stressors of different natures, including drought stress, substantially compromise the ability of plants to effectively and safely utilize light energy. We investigated the influence of water stress on the photosynthetic processes in Picea abies and Pinus sylvestris, two species with contrasting drought sensitivities. Spruce and pine seedlings were exposed to polyethylene glycol 6000-induced water deficits of different intensities and durations. The maintenance of photosystem I (PSI) oxidation in spruce required increased photosynthetic control and led to the increased reduction of the plastoquinone pool, which was not the case in pine seedlings. As a result of increased excitation pressure, photosystem II (PSII) inactivation was observed in spruce plants, whereas in pine, the decreased PSII photochemistry was likely due to sustained non-photochemical quenching. Downregulation of PSII photochemistry and maintenance of PSI in an oxidized state were linked with the prevention of oxidative stress, even under severe water deficit. The decreased photosynthetic pigment content and photosynthetic gene expression suggested the coordinated downregulation of photosynthetic apparatus components under water stress to reduce light energy absorption. In summary, the observed adaptative mechanisms of pine and spruce to water stress may be similar to the well-studied adaptative mechanisms to winter stress, which may indicate the universality of protective mechanisms under various stresses in conifers.

Comparative analysis of abscisic acid levels and expression of abscisic acid-related genes in Scots pine and Norway spruce seedlings under water deficit.

Abscisic acid (ABA) is one of the main participants in the regulation of plant responses to water deficiency. Knowledge of the ABA signal transduction pathways in gymnosperms is rather limited, especially in comparison with those in angiosperms. Seedlings of Scots pine and Norway spruce are known for their contrasting behaviour strategies under water deficit. To characterize the possible role of ABA in these differences, ABA dynamics were investigated under conditions of water deficit in seedlings of these two species. The content of ABA and its catabolites was followed in the roots and needles of seedlings of Pinus sylvestris and Picea abies under conditions of polyethylene glycol (PEG)-induced water deficiency (-0.15 and -0.5 MPa) for 10 days. The expression of the main genes for ABA-biosynthetic enzymes was also analysed. ABA showed more pronounced stress-dependent dynamics in pine roots than in spruce roots, whereas in needles, the response was greater for spruce than pine. The ABA increase during drought was mainly due to de novo synthesis and the shift in the balance between ABA synthesis and catabolism towards synthesis. The ABA-glucosyl ester did not serve as a reserve for the release of free ABA under water deficiency. The expression levels of the main ABA biosynthetic genes showed a weak or no correlation with changes in ABA content under water stress, i.e., the ABA content in the seedlings of both species was not directly linked to the transcript levels of the main ABA biosynthetic genes. Less-pronounced stress-induced changes in ABA in pine needles than in spruce needles may be related to pine seedlings having a less conservative strategy of growth and maintenance of water balance under water deficit.

Impact of UV-B radiation on the photosystem II activity, pro-/antioxidant balance and expression of light-activated genes in Arabidopsis thaliana hy4 mutants grown under light of different spectral composition.

The effect of UV-B irradiation on the photosystem II (PSII) activity, the content of photosynthetic and UV-absorbing pigments (UAPs), activity of antioxidant enzymes such as catalase (CAT) and peroxidase (POD), as well as H2O2 content in 25-day-old wild type (WT) and the cryptochrome 1 (Cry1) mutant hy4 of Arabidopsis thaliana Col-0 plants was studied. In addition, expression of photoreceptor genes Cry1, Cry2 and UVR8, photomorphogenetic gene COP1 and transcription factors genes HY5, HYH, the gene of chlorophyll-binding protein of the PSII CAB1 as well as the flavonoid biosynthesis genes CHS, PAL and thylakoid ascorbate peroxidase gene tAPX was examined. It has been shown that UV-B leads to a decrease in the photochemical activity of PSII (FV/FM) and the PSII performance index (PIABS) of WT plants grown on white (WL) and red (RL) light and also hy4 mutants grown on WL, RL and blue light (BL). In plants grown on BL and WL, the decrease in the PSII photochemical activity was significantly greater in hy4 compared to WT. The PSII of WT plants grown in BL was resistant to UV-B. The UAPs content of hy4 grown on BL and WL was lower than that in WT. The POD and CAT activities of WT grown in BL were significantly higher than in the mutant. In WT and hy4 plants grown in RL, a noticeable difference in these enzymes activity was not found. In both types of plants grown in BL and RL, the expression of photomorphogenetic genes HYH, HY5 markedly increased after UV-B treatment but the expression of the UV-B photoreceptor gene UVR8 was reduced in hy4 grown in BL and RL. It is assumed that reduced resistance of PSII in hy4 plants grown in BL and WL can be associated with low UAPs content as well as lowered POD and CAT activities. In addition, we suggest the lowered expression of UVR8 and COP1 genes caused by Cry1 deficiency leads to a shift of balance of oxidants and antioxidants towards oxidants.

Blue light alters miR167 expression and microRNA-targeted auxin response factor genes in Arabidopsis thaliana plants.

The effect of blue LED (450 nm) on the photomorphogenesis of Arabidopsis thaliana Col-0 plants and the transcript levels of several genes, including miRNAs, photoreceptors and auxin response factors (ARF) was investigated. It was observed that blue light accelerated the generative development, reduced the rosette leaf number, significantly reduced the leaf area, dry biomass and led to the disruption of conductive tissue formation. The blue LED differentially influenced the transcript levels of several phytochromes (PHY a, b, c, d, and e), cryptochromes (CRY 1 and 2) and phototropins (PHOT 1 and 2). At the same time, the blue LED significantly increased miR167 expression compared to a fluorescent lamp or white LEDs. This increase likely resulted in the enhanced transcription of the auxin response factor genes ARF4 and ARF8, which are regulated by this miRNA. These findings support the hypothesis that the effects of blue light on A. thaliana are mediated by auxin signalling pathway involving miRNA-dependent regulation of ARF gene expression.