Iron counteracts zinc-induced toxicity in soybeans.

Zinc (Zn) and iron (Fe) are essential micronutrients for all living organisms and the major targets for crop biofortification. However, when acquired in excess quantities, Zn and Fe can be toxic to plants. In this study, we examined the interaction between Zn and Fe in soybean plants under various Zn and Fe treatments. While the level of Zn accumulation increased with increasing Zn supplies, Zn content greatly decreased with rising Fe supplies. Moreover, Zn uptake rates were negatively correlated with Fe supplies. However, Fe accumulation was not greatly affected by elevating Zn supplies. Excess Zn supplies were found to induce typical Fe deficiency symptoms under low Fe conditions, which can be counteracted by increasing Fe supplies. Interestingly, leaf chlorosis caused by excess Zn and low Fe supplies was not directly associated with reduced total Fe content but likely associated with deleterious effects of excess Zn. The combination of high Zn and low Fe greatly activates FRO2 and FIT1 gene expression in soybean roots. Besides, Zn-Fe interaction influences the activities of antioxidative enzymes as well as the uptake, accumulation, and homeostasis of other essential micronutrients, such as copper and manganese in soybean plants. These findings provide new perspectives on Zn and Fe interaction and on heavy metal-induced Fe deficiency-like symptoms.

A conserved HSF:miR169:NF-YA loop involved in tomato and Arabidopsis heat stress tolerance.

Heat stress transcription factors (HSFs) and microRNAs (miRNAs) regulate different stress and developmental networks in plants. Regulatory feedback mechanisms are at the basis of these networks. Here, we report that plants improve their heat stress tolerance through HSF-mediated transcriptional regulation of MIR169 and post-transcriptional regulation of Nuclear Factor-YA (NF-YA) transcription factors. We show that HSFs recognize tomato (Solanum lycopersicum) and Arabidopsis MIR169 promoters using yeast one-hybrid/chromatin immunoprecipitation-quantitative PCR. Silencing tomato HSFs using virus-induced gene silencing (VIGS) reduced Sly-MIR169 levels and enhanced Sly-NF-YA9/A10 target expression. Further, Sly-NF-YA9/A10 VIGS knockdown tomato plants and Arabidopsis plants overexpressing At-MIR169d or At-nf-ya2 mutants showed a link with increased heat tolerance. In contrast, Arabidopsis plants overexpressing At-NF-YA2 and those expressing a non-cleavable At-NF-YA2 form (miR169d-resistant At-NF-YA2) as well as plants in which At-miR169d regulation is inhibited (miR169d mimic plants) were more sensitive to heat stress, highlighting NF-YA as a negative regulator of heat tolerance. Furthermore, post-transcriptional cleavage of NF-YA by elevated miR169 levels resulted in alleviation of the repression of the heat stress effector HSFA7 in tomato and Arabidopsis, revealing a retroactive control of HSFs by the miR169:NF-YA node. Hence, a regulatory feedback loop involving HSFs, miR169s and NF-YAs plays a critical role in the regulation of the heat stress response in tomato and Arabidopsis plants.

Novel insights into expansion and functional diversification of MIR169 family in tomato.

MAIN CONCLUSION: Expansion of MIR169 members by duplication and new mature forms, acquisition of new promoters, differential precursor-miRNA processivity and engaging novel targets increase the functional diversification of MIR169 in tomato. MIR169 family is an evolutionarily conserved miRNA family in plants. A systematic in-depth analysis of MIR169 family in tomato is lacking. We report 18 miR169 precursors, annotating new loci for MIR169a, b and d, as well as 3 novel mature isoforms (MIR169f/g/h). The family has expanded by both tandem- and segmental-duplication events during evolution. A tandem-pair MIR169b/b-1 and MIR169b-2/h is polycistronic in nature coding for three MIR169b isoforms and a new variant miR169h, that is evidently absent in the wild relatives S. pennellii and S. pimpinellifolium. Seven novel miR169 targets including RNA-binding protein, protein-phosphatase, aminotransferase, chaperone, tetratricopeptide-repeat-protein, and transcription factors ARF-9B and SEPELLATA-3 were established by efficient target cleavage in the presence of specific precursors as well as increased target abundance upon miR169 chelation by short-tandem-target-mimic construct in transient assays. Comparative antagonistic expression profiles of MIR169:target pairs suggest MIR169 family as ubiquitous regulator of various abiotic stresses (heat, cold, dehydration and salt) and developmental pathways. This regulation is partly brought about by acquisition of new promoters as demonstrated by promoter MIR169:GUS reporter assays as well as differential processivity of different precursors and miRNA cleavage efficiencies. Thus, the current study augments the functional horizon of MIR169 family with applications for stress tolerance in crops.