Distribution and Redistribution of 109Cd and 65Zn in the Heavy Metal Hyperaccumulator Solanum nigrum L.: Influence of Cadmium and Zinc Concentrations in the Root Medium.

Heavy metal redistribution is relevant for the quality of edible crops and the suitability of hyperaccumulators for bioremediation. Root-to-shoot transfer via the xylem and redistribution in the aerial parts via the phloem differ between various heavy metals. In general, cadmium is more slowly released to the shoot than zinc (e.g., in wheat, bean, and lupin). However, rapid cadmium transport to the shoot was detected in the hyperaccumulator Solanum nigrum L. This is a key aspect in this article and might be important for bioremediation. The radionuclides 109Cd and 65Zn were used to investigate the respective influence of elevated cadmium or zinc in the root medium on the dynamics of the two heavy metals in S. nigrum. Although transport via the xylem to the leaves was similar for 109Cd and 65Zn, the further redistribution from older leaves to younger leaves, flowers, and fruits via the phloem was far less efficient for 109Cd than for 65Zn. Furthermore, the redistribution of 109Cd within the shoot was negatively influenced by increased cadmium (but not by increased zinc) concentrations in the nutrient medium. The redistribution of 65Zn in the shoot was selectively decreased by increased zinc concentrations (but generally not by cadmium).

Effects of PEG-Induced Water Deficit in Solanum nigrum on Zn and Ni Uptake and Translocation in Split Root Systems.

Drought strongly influences root activities in crop plants and weeds. This paper is focused on the performance of the heavy metal accumulator Solanum nigrum, a plant which might be helpful for phytoremediation. The water potential in a split root system was decreased by the addition of polyethylene glycol (PEG 6000). Rubidium, strontium and radionuclides of heavy metals were used as markers to investigate the uptake into roots, the release to the shoot via the xylem, and finally the basipetal transport via the phloem to unlabeled roots. The uptake into the roots (total contents in the plant) was for most makers more severely decreased than the transport to the shoot or the export from the shoot to the unlabeled roots via the phloem. Regardless of the water potential in the labeling solution, (63)Ni and (65)Zn were selectively redistributed within the plant. From autoradiographs, it became evident that (65)Zn accumulated in root tips, in the apical shoot meristem and in axillary buds, while (63)Ni accumulated in young expanded leaves and roots but not in the meristems. Since both radionuclides are mobile in the phloem and are, therefore, well redistributed within the plant, the unequal transfer to shoot and root apical meristems is most likely caused by differences in the cell-to-cell transport in differentiation zones without functional phloem (immature sieve tubes).

Selective uptake, distribution, and redistribution of (109)Cd, (57)Co, (65)Zn, (63)Ni, and (134)Cs via xylem and phloem in the heavy metal hyperaccumulator Solanum nigrum L.

The focus of this article was to explore the translocation of (109)Cd, (57)Co, (65)Zn, (63)Ni, and (134)Cs via xylem and phloem in the newly found hyperaccumulator Solanum nigrum L. Two experiments with the uptake via the roots and transport of (109)Cd, (57)Co, and (65)Zn labeled by roots, and the redistribution of (109)Cd, (65)Zn, (57)Co, (63)Ni, and (134)Cs using flap label in S. nigrum in a hydroponic culture with a standard nutrient solution were conducted. The results showed that (109)Cd added for 24 h to the nutrient medium of young plants was rapidly taken up, transferred to the shoot, and accumulated in the cotyledons and the oldest leaves but was not efficiently redistributed within the shoot afterward leading to a rather low content in the fruits. In contrast, (57)Co was more slowly taken up and released to the shoot, but afterward, this element was redistributed from older leaves to younger leaves and maturing fruits. (65)Zn was rapidly taken up and transferred to the shoot (mainly to the youngest leaves and not to the cotyledons). Afterward, this radionuclide was redistributed within the shoot to the youngest organs and finally accumulated in the maturing fruits. After flap labeling, all five heavy metals tested ((109)Cd, (57)Co, (65)Zn, (63)Ni, (134)Cs) were exported from the labeled leaf and redistributed within the plant. The accumulation in the fruits was most pronounced for (63)Ni and (65)Zn, while a relatively high percentage of (57)Co was finally found in the roots. (134)Cs was roughly in the middle of them. The transport of (109)Cd differed from that previously reported for wheat or lupin and might be important for the potential of S. nigrum to hyperaccumulate cadmium.

Dehydrin expression as a potential diagnostic tool for cold stress in white clover.

Cold acclimation is important for crop survival in environments undergoing seasonal low temperatures. It involves the induction of defensive mechanisms including the accumulation of different cryoprotective molecules among which are dehydrins (DHN). Recently several sequences coding for dehydrins were identified in white clover (Trifolium repens). This work aimed to select the most responsive to cold stress DHN analogues in search for cold stress diagnostic markers. The assessment of dehydrin transcript accumulation via RT-PCR and immunodetection performed with three antibodies against the conserved K-, Y-, and S-segment allowed to outline different dehydrin types presented in the tested samples. Both analyses confirmed that YnKn dehydrins were underrepresented in the controls but exposure to low temperature specifically induced their accumulation. Strong immunosignals corresponding to 37-40 kDa with antibodies against Y- and K-segment were revealed in cold-stressed leaves. Another 'cold-specific' band at position 52-55 kDa was documented on membranes probed with antibodies against K-segment. Real time RT-qPCR confirmed that low temperatures induced the accumulation of SKn and YnSKn transcripts in leaves and reduced their expression in roots. Results suggest that a YnKn dehydrin transcript with GenBank ID: KC247805 and the immunosignal at 37-40 kDa, obtained with antibodies against Y- and K-segment are reliable markers for cold stress in white clover. The assessment of SKn (GenBank ID: EU846208) and YnSKn (GenBank ID: KC247804) transcript levels in leaves could serve as additional diagnostic tools.

Identification and expression of different dehydrin subclasses involved in the drought response of Trifolium repens.

Reverse transcribed RNAs coding for YnKn, YnSKn, SKn, and KS dehydrin types in drought-stressed white clover (Trifolium repens) were identified and characterized. The nucleotide analyses revealed the complex nature of dehydrin-coding sequences, often featured with alternative start and stop codons within the open reading frames, which could be a prerequisite for high variability among the transcripts originating from a single gene. For some dehydrin sequences, the existence of natural antisense transcripts was predicted. The differential distribution of dehydrin homologues in roots and leaves from a single white clover stolon under normal and drought conditions was evaluated by semi-quantitative RT-PCR and immunoblots with antibodies against the conserved K-, Y- and S-segments. The data suggest that different dehydrin classes have distinct roles in the drought stress response and vegetative development, demonstrating some specific characteristic features. Substantial levels of YSK-type proteins with different molecular weights were immunodetected in the non-stressed developing leaves. The acidic SK2 and KS dehydrin transcripts exhibited some developmental gradient in leaves. A strong increase of YK transcripts was documented in the fully expanded leaves and roots of drought-stressed individuals. The immunodetected drought-induced signals imply that Y- and K-segment containing dehydrins could be the major inducible Late Embryogenesis Abundant class 2 proteins (LEA 2) that accumulate predominantly under drought.

Genotypic variation in drought stress response and subsequent recovery of wheat (Triticum aestivum L.).

Three wheat (Triticum aestivum L.) genotypes, Sadovo, Katya and Prelom, with different tolerance to drought were comparatively evaluated in terms of leaf respiratory responses to progressing dehydration and consecutive rewatering. Under drought stress, the respiration of all varieties gradually decreased, as the drought-tolerant Katya showed the most pronounced decline at earlier stages of dehydration. When water stress intensified, this genotype gave relatively stable respiration rates compared with the drought-sensitive varieties. Additionally, dehydrated Katya leaves displayed lower stomatal conductance and higher photosynthesis values, which resulted in greater water use efficiency during the dehydration period. Combination of drought stress and short-term changes in leaf temperature also induced genotype-specific response that differed from the response to drought only. Over the whole temperature range, the leaves of Katya exposed to dehydration for 14 days, showed higher respiration rates compared to the drought-sensitive varieties. The sensitive varieties maintained higher respiration rates under control conditions and mild dehydration, and very low rates under severe drought. In Katya, respiration and photosynthesis were fully restored from the stress within the first day of rewatering. The drought-sensitive genotypes displayed a considerably slower recovering capacity. The results are discussed in terms of possible physiological mechanisms underlying plant tolerance to drought.

Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant protein in photosynthesizing plant parts and the most abundant protein on earth. Amino acids deriving from its net degradation during senescence are transported to sinks (e.g. developing leaves, fruits). Rubisco catabolism is not controlled only by the overall sink demand. An accumulation of carbohydrates may also accelerate senescence and Rubisco degradation under certain conditions. Amino acids produced by proteolysis are rapidly redistributed in plants with proper source-sink relationships. In leaves of wheat plants with reduced sink capacity (e.g. sink removal, phloem interruption by steam girdling at the leaf base), Rubisco is degraded and free amino acids accumulate. They may be washed out in the rain during late senescence. In leaves of depodded soybeans, Rubisco is degraded and amino acids can be reutilized in these leaves for the synthesis of special vacuolar proteins in the paraveinal mesophyll (vegetative storage proteins). Nitrogen deriving from Rubisco degradation in older (senescing) leaves of annual crops is integrated to some extent again in newly synthesized Rubisco in younger leaves or photosynthesizing tissues of fruits. Finally, a high percentage of this nitrogen is accumulated in protein bodies (storage proteins). At the subcellular level, Rubisco can be degraded in intact chloroplasts. Reactive oxygen species may directly cleave the large subunit or modify it to become more susceptible to proteolysis. A metalloendopeptidase may play an important role in Rubisco degradation within intact chloroplasts. Additionally, the involvement of vacuolar endopeptidase(s) in Rubisco catabolism (at least under certain conditions) was postulated by various laboratories.

In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown.

A central reaction of chlorophyll breakdown, porphyrin ring opening of pheophorbide a to the primary fluorescent chlorophyll catabolite (pFCC), requires pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), with red chlorophyll catabolite (RCC) as a presumably PAO-bound intermediate. In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). Here, we show that RCCR-deficient Arabidopsis thaliana accumulates RCC and three RCC-like pigments during senescence, as well as FCCs and NCCs. We also show that the stereospecificity of Arabidopsis RCCR is defined by a small protein domain and can be reversed by a single Phe-to-Val exchange. Exploiting this feature, we prove the in vivo participation of RCCR in chlorophyll breakdown. After complementation of RCCR mutants with RCCRs exhibiting alternative specificities, patterns of chlorophyll catabolites followed the specificity of complementing RCCRs. Light-dependent leaf cell death observed in different RCCR-deficient lines strictly correlated with the accumulation of RCCs and the release of singlet oxygen, and PAO induction preceded lesion formation. These findings suggest that RCCR absence causes leaf cell death as a result of the accumulation of photodynamic RCC. We conclude that RCCR (together with PAO) is required for the detoxification of chlorophyll catabolites and discuss the biochemical role(s) for this enzyme.

Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction.

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age- and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.

Chlorophyll breakdown: pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene.

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron-sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.