Spatiotemporal heterogeneity of photosystem II function during acclimation to zinc exposure and mineral nutrition changes in the hyperaccumulator Noccaea caerulescens.

We investigated changes in mineral nutrient uptake and translocation and photosystem II (PSII) functionality, in the hyperaccumulator Noccaea caerulescens after exposure to 800 µM Zn in hydroponic culture. Exposure to Zn inhibited the uptake of K, Mn, Cu, Ca, and Mg, while the uptake of Fe and Zn enhanced. Yet, Ca and Mg aboveground tissue concentrations remain unchanged while Cu increased significantly. In the present study, we provide new data on the mechanism of N. caerulescens acclimation to Zn exposure by elucidating the process of photosynthetic acclimation. A spatial heterogeneity in PSII functionality in N. caerulescens leaves exposed to Zn for 3 days was detected, while a threshold time of 4 days was needed for the activation of Zn detoxification mechanism(s) to decrease Zn toxicity and for the stomatal closure to decrease Zn supply at the severely affected leaf area. After 10-day exposure to Zn, the allocation of absorbed light energy in PSII under low light did not differ compared to control ones, while under high light, the quantum yield of non-regulated energy loss in PSII (ΦNO) was lower than the control, due to an efficient photoprotective mechanism. The chlorophyll fluorescence images of non-photochemical quenching (NPQ) and photochemical quenching (qp) clearly showed spatial and temporal heterogeneity in N. caerulescens exposure to Zn and provided further information on the particular leaf area that was most sensitive to heavy metal stress. We propose the use of chlorophyll fluorescence imaging, and in particular the redox state of the plastoquinone (PQ) pool that was found to display the highest spatiotemporal heterogeneity, as a sensitive bio-indicator to measure the environmental pressure by heavy metals on plants.

Cadmium-zinc accumulation and photosystem II responses of Noccaea caerulescens to Cd and Zn exposure.

A population of the metallophyte Noccaea (Thlaspi) caerulescens originating from a Zn-enriched area at Røros Copper Mine (Norway) was studied. N. caerulescens tolerance to accumulate Cd and Zn was evaluated in hydroponic experiments by chlorophyll fluorescence imaging analysis. In the field-collected N. caerulescens mother plants, Zn shoot concentrations were above Zn hyperaccumulation threshold while, in hydroponic experiments under 40-µM Cd exposure, shoot Cd concentrations were clearly above Cd hyperaccumulation threshold. Cadmium ions and, to a less extent, Zn were mainly retained in the roots. Exposure to Cd enhanced Zn translocation to the shoot, while decreased significant total Ca2+ uptake, suggesting that Cd uptake occurs through Ca2+ transporters. Nevertheless, it increased Ca2+ translocation to the leaf, possibly for photoprotection of photosystem II (PSII). Exposure to 800 µM Zn or 40 µM Cd resulted in increased Fe3+ uptake suggesting that in N. caerulescens, Cd uptake does not take place through the pathway of Fe3+ uptake and that conditions that lead to Cd and Zn accumulation in plants may also favor Fe accumulation. Despite the significant high toxicity levels of Zn and Cd in leaves, under Zn and Cd exposure, respectively, the allocation of absorbed light energy at PSII did not differ compared to controls. The results showed that N. caerulescens keep Cd and Zn concentrations in the mesophyll cells in non-toxic forms for PSII and that the increased Ca and Fe accumulation in leaves alleviates the toxicity effects. Chlorophyll fluorescence imaging revealed that PSII of N. caerulescens resisted better the phytotoxic effects of 20 times higher Zn than Cd exposure concentration. Overall, it is concluded that the use of chlorophyll fluorescence imaging constitutes a promising basis for investigating heavy metal tolerance of plants.

Regulatory mechanisms after short- and long-term perturbed lysine biosynthesis in the aspartate pathway: the need for isogenes in Arabidopsis thaliana.

The aspartate-derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.2.4), homoserine dehydrogenase (HSDH; EC 1.1.1.3) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7), formerly referred to as dihydrodipicolinate synthase (DHDPS). They are encoded by isoenzyme families and it is not known why such families are evolutionarily maintained. To gain more insight into the specific roles and regulation of the isoenzymes, we inhibited DHDPS in Arabidopsis thaliana with the chemical compound (N,N-dimethylglycinatoboranyloxycarbonylmethyl)-dimethylamine-borane (DDAB) and compared the short-term effects on the biochemical and biomolecular level to the long-term adaptations in dhdps knockout mutants. We found that DHDPS2 plays a crucial role in controlling lysine biosynthesis, thereby stabilizing flux through the whole aspartate pathway. Moreover, DHDPS2 was also shown to influence the threonine level to a large extent. In addition, the lysine-sensitive AKs, AKLYS1 and AKLYS3 control the short- and long-term responses to perturbed lysine biosynthesis in Arabidopsis thaliana.

Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant.

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.