FTSH4 and OMA1 mitochondrial proteases reduce moderate heat stress-induced protein aggregation.

The threat of global warming makes uncovering mechanisms of plant tolerance to long-term moderate heat stress particularly important. We previously reported that Arabidopsis (Arabidopsis thaliana) plants lacking mitochondrial proteases FTSH4 or OMA1 suffer phenotypic changes under long-term stress of 30°C, while their growth at 22°C is not affected. Here we found that these morphological and developmental changes are associated with increased accumulation of insoluble mitochondrial protein aggregates that consist mainly of small heat-shock proteins (sHSPs). Greater accumulation of sHSPs in ftsh4 than oma1 corresponds with more severe phenotypic abnormalities. We showed that the proteolytic activity of FTSH4, and to a lesser extent of OMA1, as well as the chaperone function of FTSH4, is crucial for protecting mitochondrial proteins against aggregation. We demonstrated that HSP23.6 and NADH dehydrogenase subunit 9 present in aggregates are proteolytic substrates of FTSH4, and this form of HSP23.6 is also a substrate of OMA1 protease. In addition, we found that the activity of FTSH4 plays an important role during recovery from elevated to optimal temperatures. Isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analyses, along with identification of aggregation-prone proteins, implicated mitochondrial pathways affected by protein aggregation (e.g. assembly of complex I) and revealed that the mitochondrial proteomes of ftsh4 and oma1 plants are similarly adapted to long-term moderate heat stress. Overall, our data indicate that both FTSH4 and OMA1 increase the tolerance of plants to long-term moderate heat stress by reducing detergent-tolerant mitochondrial protein aggregation.

NO-Dependent programmed cell death is involved in the formation of Zn-related lesions in tobacco leaves.

A recent study indicated that the development of lesions on the leaf blades of tobacco exposed to zinc (Zn) excess can be considered a manifestation of a Zn-tolerance strategy at the organ level. Here, we investigated whether cell death leading to the formation of localized lesions is destructive in character (necrosis type) or results from programmed self-induced cell death (PCD). Selected parameters, including PCD markers, were determined in the leaves from tobacco plants grown in the presence of 200 µM Zn and compared with control conditions. TUNEL assay results showing internucleosomal DNA fragmentation in the nuclei of the cells from Zn-exposed leaves, together with an enhanced expression of three PCD marker genes (NtBI-1, Ntrboh, and NtSIPK), indicated the involvement of PCD in the formation of Zn-related lesions. It is known that NO is a key factor in the execution of PCD. Interestingly, upon exposure to high Zn, in situ localization of NO (visualized using DAF-2DA fluorescence) was restricted to groups of mesophyll cells, and was correlated with the pattern of Zn localization (determined using the fluorophore Zinpyr-1), similarly limited primarily to groups of "Zn accumulating cells". Furthermore, inhibition of the formation of lesions in the presence of l-NAME (an NO synthase inhibitor) was accompanied by the delayed appearance of Zn and by NO localization limited to these groups of cells. Altogether, we provide the first demonstration that Zn-related lesions in leaves develop from groups of mesophyll cells in which accumulation of high concentrations of Zn contributes to enhancement of the NO level and to initiation of PCD processes.

The ratio of Zn to Cd supply as a determinant of metal-homeostasis gene expression in tobacco and its modulation by overexpressing the metal exporter AtHMA4.

This study links changes in the tobacco endogenous metal-homeostasis network caused by transgene expression with engineering of novel features. It also provides insight into the concentration-dependent mutual interactions between Zn and Cd, leading to differences in the metal partitioning between wild-type and transgenic plants. In tobacco, expression of the export protein AtHMA4 modified Zn/Cd root/shoot distribution, but the pattern depended on their concentrations in the medium. To address this phenomenon, the expression of genes identified by suppression subtractive hybridization and the Zn/Cd accumulation pattern were examined upon exposure to six variants of low/high Zn and Cd concentrations. Five tobacco metal-homeostasis genes were identified: NtZIP2, NtZIP4, NtIRT1-like, NtNAS, and NtVTL. In the wild type, their expression depended on combinations of low/high Zn and Cd concentrations; co-ordinated responses of NtZIP1, NtZIP2, and NtVTL were shown in medium containing 4 µM Cd, and at 0.5 µM versus 10 µM Zn. In transgenics, qualitative changes detected for NtZIP1, NtZIP4, NtIRT1-like, and NtVTL are considered crucial for modification of Zn/Cd supply-dependent Zn/Cd root/shoot distribution. Notwithstanding, NtVTL was the most responsive gene in wild-type and transgenic plants under all concentrations of Zn and Cd tested; thus it is a candidate gene for the regulation of metal cross-homeostasis processes involved in engineering new metal-related traits.

Determination the Usefulness of AhHMA4p1::AhHMA4 Expression in Biofortification Strategies.

AhHMA4 from Arabidopsis thaliana encodes Zn/Cd export protein that controls Zn/Cd translocation to shoots. The focus of this manuscript is the evaluation of AhHMA4 expression in tomato for mineral biofortification (more Zn and less Cd in shoots and fruits). Hydroponic and soil-based experiments were performed. Transgenic and wild-type plants were grown on two dilution levels of Knop's medium (1/10, 1/2) with or without Cd, to determine if mineral composition affects the pattern of root/shoot partitioning of both metals due to AhHMA4 expression. Facilitation of Zn translocation to shoots of 19-day-old transgenic tomato was noted only when plants were grown in the more diluted medium. Moreover, the expression pattern of Zn-Cd-Fe cross-homeostasis genes (LeIRT1, LeChln, LeNRAMP1) was changed in transgenics in a medium composition-dependent fashion. In plants grown in soil (with/without Cd) up to maturity, expression of AhHMA4 resulted in more efficient translocation of Zn to shoots and restriction of Cd. These results indicate the usefulness of AhHMA4 expression to improve the growth of tomato on low-Zn soil, also contaminated with Cd.

Development of Zn-related necrosis in tobacco is enhanced by expressing AtHMA4 and depends on the apoplastic Zn levels.

AtHMA4 was previously shown to contribute to the control of Zn root-to-shoot translocation and tolerance to high Zn. However, heterologous expression of 35S::AtHMA4 in tobacco (Nicotiana tabacum cv. Xanthi) results in enhanced Zn sensitivity. This study provides a better understanding of the development of this Zn-sensitive phenotype and demonstrates that substantial modifications of Zn homeostasis occur due to AtHMA4 expression. We show that ectopically expressing AtHMA4 in tobacco results in overloading the root and leaf apoplast with Zn. The tissue and cellular distribution of Zn, monitored using Zinpyr-1, was altered in the AtHMA4-expressing plants compared with wild type. Increased loading of the leaf apoplast with Zn in AtHMA4 transformants induced necrosis; this appeared at lower levels of Zn supply in the transgenics compared with wild type. This study suggests that Zn concentration may be sensed in the apoplast of leaves, and if concentrations are above a certain threshold then particular groups of cells accumulate Zn and necrosis is initiated. Therefore, this could be considered as a mechanism for protecting the other parts of the photosynthetically active leaf from Zn toxicity.