Genome-wide analysis of the MADS-box gene family of sea buckthorn (Hippophae rhamnoides ssp. sinensis) and their potential role in floral organ development.

Sea buckthorn (Hippophae rhamnoides ssp. sinensis) is a deciduous shrub or small tree in the Elaeagnaceae family. It is dioecious, featuring distinct structures in female and male flowers. The MADS-box gene family plays a crucial role in flower development and differentiation of floral organs in plants. However, systematic information on the MADS-box family in sea buckthorn is currently lacking. This study presents a genome-wide survey and expression profile of the MADS-box family of sea buckthorn. We identified 92 MADS-box genes in the H. rhamnoides ssp. Sinensis genome. These genes are distributed across 12 chromosomes and classified into Type I (42 genes) and Type II (50 genes). Based on the FPKM values in the transcriptome data, the expression profiles of HrMADS genes in male and female flowers of sea buckthorn showed that most Type II genes had higher expression levels than Type I genes. This suggesting that Type II HrMADS may play a more significant role in sea buckthorn flower development. Using the phylogenetic relationship between sea buckthorn and Arabidopsis thaliana, the ABCDE model genes of sea buckthorn were identified and some ABCDE model-related genes were selected for qRT-PCR analysis in sea buckthorn flowers and floral organs. Four B-type genes may be involved in the identity determination of floral organs in male flowers, and D-type genes may be involved in pistil development. It is hypothesized that ABCDE model genes may play an important role in the identity of sea buckthorn floral organs. This study analyzed the role of MADS-box gene family in the development of flower organs in sea buckthorn, which provides an important theoretical basis for understanding the regulatory mechanism of sex differentiation in sea buckthorn.

MTP8 from Triticum urartu Is Primarily Responsible for Manganese Tolerance.

Mineral nutrients, such as manganese (Mn) and iron (Fe), play essential roles in many biological processes in plants but their over-enrichment is harmful for the metabolism. Metal tolerance proteins (MTPs) are involved in cellular Mn and Fe homeostasis. However, the transporter responsible for the transport of Mn in wheat is unknown. In our study, TuMTP8, a Mn-CDF transporter from diploid wheat (Triticum urartu), was identified. Expression of TuMTP8 in yeast strains of Δccc1 and Δsmf1 and Arabidopsis conferred tolerance to elevated Mn and Fe, but not to other metals (zinc, cobalt, copper, nickel, or cadmium). Compared with TuVIT1 (vacuole Fe transporter), TuMTP8 shows a significantly higher proportion in Mn transport and a smaller proportion in Fe transport. The transient analysis in tobacco epidermal cells revealed that TuMTP8 localizes to vacuolar membrane. The highest transcript levels of TuMTP8 were in the sheath of the oldest leaf and the awn, suggesting that TuMTP8 sequesters excess Mn into the vacuole in these organs, away from more sensitive tissues. These findings indicate that TuMTP8, a tonoplast-localized Mn/Fe transporter, functions as a primary balancer to regulate Mn distribution in T. urartu under elevated Mn conditions and participates in the intracellular transport and storage of excess Mn as a detoxification mechanism, thereby conferring Mn tolerance.

Characterization of the gene family encoding metal tolerance proteins in Triticum urartu: Phylogenetic, transcriptional, and functional analyses.

Homeostasis of microelements in organisms is vital for normal metabolism. In plants, the cation diffusion facilitator (CDF) protein family, also known as metal tolerance proteins (MTPs), play critical roles in maintaining trace metal homeostasis. However, little is known about these proteins in wheat. In this study, we characterized the MTP family of Triticum urartu, the donor of 'A' genome of Triticum aestivum, and analysed their phylogenetic relationships, sequence signatures, spatial expression patterns in the diploid wheat, and their transport activity when heterologously expressed. Nine MTPs were identified in the T. urartu genome database, and were classified and designated based on their sequence similarity to Arabidopsis thaliana (Arabidopsis) and Oryza sativa MTPs. Phylogenetic and sequence analyses indicated that the triticum urartu metal tolerance protein (TuMTP)s comprise three Zn-CDFs, two Fe/Zn-CDFs, and four Mn-CDFs; and can be further classified into six subgroups. Among the TuMTPs, there are no MTP2-5 and MTP9-10 counterparts but two MTP1/8/11 orthologs in relation to AtMTPs. It was also shown that members of the same cluster share similar sequence characteristic, i.e. number of introns, predicted transmembrane domains, and motifs. When expressed in yeast, TuMTP1 and TuMTP1.1 conferred tolerance to Zn and Co but not to other metal ions; while TuMTP8, TuMTP8.1, TuMTP11, and TuMTP11.1 conferred tolerance to Mn. When expressed in Arabidopsis, TuMTP1 localized to the tonoplast and significantly enhanced Zn and Co tolerance. TuMTPs showed diverse tissue-specific expression patterns. Taken together, the closely clustered TuMTPs share structural features and metal specificity but play diverse roles in the homeostasis of microelements in plant cells.

New Biofortification Tool: Wheat TaCNR5 Enhances Zinc and Manganese Tolerance and Increases Zinc and Manganese Accumulation in Rice Grains.

Heavy metal contaminants and nutrient deficiencies in soil negatively affect crop growth and human health. The plant cadmium resistance (PCR) protein transports heavy metals. The abundance of PCR is correlated with that of cell number regulator (CNR) protein, and the two proteins have similar conserved domains. Hence, CNR might also participate in heavy metal transport. We isolated and analyzed TaCNR5 from wheat (Triticum aestivum). The expression level of TaCNR5 in the shoots of wheat increased under cadmium (Cd), zinc (Zn), or manganese (Mn) treatments. Transgenic plants expressing TaCNR5 showed enhanced tolerance to Zn and Mn. Overexpression of TaCNR5 in Arabidopsis increased Cd, Zn, and Mn translocation from roots to shoots. The concentrations of Zn and Mn in rice grains were increased in transgenic plants expressing TaCNR5. These roles of TaCNR5 in the translocation and distribution of heavy metals mean that it has potential as a genetic biofortification tool to fortify cereal grains with micronutrients.

Co-expression of multiple heavy metal transporters changes the translocation, accumulation, and potential oxidative stress of Cd and Zn in rice (Oryza sativa).

The OsHMA2, OsLCT1 and OsZIP3 transporters were all involved in zinc (Zn) and cadmium (Cd) transport. So far, only a few researches studied on the co-regulation effect of three transporters related to Zn and Cd transport. The present study showed that rice co-expressing OsLCT1-OsHMA2-OsZIP3 (LHZ) had longer roots and shoots than wild-type (WT) rice after Zn and Cd treatments. The chlorophyll content was significantly higher, and the proline, malondialdehyde and H2O2 contents were significantly lower in co-transgenic lines than in WT under Cd and Zn stress. LHZ in the seedlings of transgenic rice decreased the root-to-shoot translocation of Cd after Cd and Zn treatments. At the filling stage, LHZ line reduced Cd accumulation in grain after Cd treatment. Moreover, LHZ line increased the translocation of Zn to grain and reduced the accumulation of Cd after Zn treatment. These results suggested that LHZ co-expression could effectively decrease the translocation and accumulation of Cd to grains, alleviated the oxidative stress of Cd and Zn, and finally enhanced the quality and safety of rice grains.

Effects of cadmium toxicity on diploid wheat (Triticum urartu) and the molecular mechanism of the cadmium response.

Cadmium (Cd) is a widespread soil contaminant that readily accumulates in wheat, and posing a potential threat to human health. Our aim is to investigate Cd toxicity effect and molecular mechanisms for wheat. In this study, the physiological indexes, morphology, and gene expression patterns of diploid wheat (Triticum urartu) seedlings were evaluated after 2 and 5 d of a Cd treatment (10 µM CdSO4). The Cd treatment resulted in increased proline and glutathione contents in shoots and roots, slight damage to leaf tips, severe damage to root tips, and increased root secretions. Transcriptome analysis showed that there were significantly more differentially expressed genes (DEGs) in shoots and roots after 5 d of Cd stress than after 2 d of Cd stress, and the DEGs of the shoots were more different than the roots. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the pathways enriched under Cd treatment were "DNA replication" and "phenylpropanoid biosynthesis". These findings provide information about the responses to Cd stress in wheat, and provide a theoretical basis for reducing Cd toxicity and protecting food safety.

Heterologous expression of TuCAX1a and TuCAX1b enhances Ca2+ and Zn2+ translocation in Arabidopsis.

KEY MESSAGE: TuCAX1a and TuCAX1b improved Ca2+ and Zn2+ translocation and TuCAX1b enhanced Ca2+, Zn2+, Mn2+ and Fe2+ content when exposed to Cd2+; Cd2+ translocation was inhibited under Ca2+ and Zn2+. Cation/H+ antiporters (CAXs) are involved in the translocation of Ca2+ and various metal ions in higher plants. In the present study, TuCAX1a and TuCAX1b, two cation/H+ antiporters, were isolated from the diploid wheat Triticum urartu, and their metal cation translocation functions investigated. TuCAX1a and TuCAX1b showed abundant tissue-specific expression in the internode and beard, respectively, and their expression levels were increased in shoots exposed to Cd2+, Zn2+ and Ca2+. Plant phenotype analysis showed that overexpression of TuCAX1a and TuCAX1b could improve the tolerance of Arabidopsis to exogenous Ca2+ and Zn2+. In the plant shoots and roots, the contents of Ca2+ and Zn2+ were higher than wild-type plants under Ca2+ and Zn2+ treatments, indicating that TuCAX1a and TuCAX1b can enhance Ca2+ and Zn2+ translocation. Ca2+, Zn2+, Mn2+ and Fe2+ contents showed higher accumulation in TuCAX1b-transgenic Arabidopsis shoots than in wild-type plants exposed to Cd2+, and the translocation of Cd2+ was inhibited under Ca2+ and Zn2+. Overall, the present study provides a novel genetic resource for improving the uptake of microelements and reducing accumulation of toxic heavy metals in wheat.

Improved Cd, Zn and Mn tolerance and reduced Cd accumulation in grains with wheat-based cell number regulator TaCNR2.

Soil microelement deficiency and heavy metal contamination affects plant growth and development, but improving trace element uptake and reducing heavy metal accumulation by genetic breeding can help alleviate this. Cell number regulator 2 (TaCNR2) from common wheat (Triticum aestivum) are similar to plant cadmium resistance proteins, involved with regulating heavy metal translocation. Our aim was to understand the effect of TaCNR2 on heavy metal tolerance and translocation. In this study, real-time quantitative PCR indicated TaCNR2 expression in the wheat seedlings increased under Cd, Zn and Mn treatment. Overexpression of TaCNR2 in Arabidopsis and rice enhanced its stress tolerance to Cd, Zn and Mn, and overexpression in rice improved Cd, Zn and Mn translocation from roots to shoots. The grain husks in overexpressed rice had higher Cd, Zn and Mn concentrations, but the brown rice accumulated less Cd but higher Mn than wild rice. The results showed that TaCNR2 can transport heavy metal ions. Thus, this study provides a novel gene resource for increasing nutrition uptake and reducing toxic metal accumulation in crops.

Triticum urartu MTP1: its ability to maintain Zn2+ and Co2+ homeostasis and metal selectivity determinants.

KEY MESSAGE: TuMTP1 maintains Zn2+ and Co2+ homeostasis by sequestering excess Zn2+ and Co2+ into vacuoles. The mutations NSEDD/VTVTT in the His-rich loop and I119F in TMD3 of TuMTP1 restrict metal selectivity. Mineral nutrients, such as zinc (Zn) and cobalt (Co), are essential or beneficial for plants but can be toxic at elevated levels. Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. However, the determinants of substrate selectivity have not been clarified due to the diversity of MTP1 substrates in various plants. In this study, Triticum urartu MTP1 was characterized. When expressed in yeast, TuMTP1 conferred tolerance to Zn2+ and Co2+ but not Fe2+, Cu2+, Ni2+ or Cd2+ in solid and liquid culture and localized on the vacuolar membrane. Furthermore, TuMTP1-expressing yeast accumulated more Zn2+ and Co2+ when treated. TuMTP1 expression in T. urartu roots was significantly increased under Zn2+ and Co2+ stresses. Determinants of substrate selectivity were then examined through site-directed mutagenesis. The exchange of NSEDD with VTVTT in the His-rich loop of TuMTP1 restricted its metal selectivity to Zn2+, whereas the I119F mutation confined specificity to Co2+. The mutations H74, D78, H268 and D272 (in the Zn2+-binding site) and Leu322 (in the C-terminal Leu-zipper) partially or completely abolished the transport function of TuMTP1. These results show that TuMTP1 might sequester excess cytosolic Zn2+ and Co2+ into yeast vacuoles to maintain Zn2+ and Co2+ homeostasis. The NSEDD/VTVTT and I119F mutations are crucially important for restricting the substrate specificity of TuMTP1, and the Zn2+-binding site and Leu322 are essential for its ion selectivity and transport function. These results can be employed to change metal selectivity for biofortification or phytoremediation applications.

Applying DNA Barcodes to Identify Closely Related Species of Ferns: A Case Study of the Chinese Adiantum (Pteridaceae).

DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions (rbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight nuclear regions (ITS, pgiC, gapC, LEAFY, ITS2, IBR3_2, DET1, and SQD1_1) were screened and evaluated in the fern genus Adiantum from China and neighboring areas. Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure.