Identification and Functional Characterization of Abiotic Stress Tolerance-Related PLATZ Transcription Factor Family in Barley (Hordeum vulgare L.).

Plant AT-rich sequence and zinc-binding proteins (PLATZs) are a novel category of plant-specific transcription factors involved in growth, development, and abiotic stress responses. However, the PLATZ gene family has not been identified in barley. In this study, a total of 11 HvPLATZs were identified in barley, and they were unevenly distributed on five of the seven chromosomes. The phylogenetic tree, incorporating PLATZs from Arabidopsis, rice, maize, wheat, and barley, could be classified into six clusters, in which HvPLATZs are absent in Cluster VI. HvPLATZs exhibited conserved motif arrangements with a characteristic PLATZ domain. Two segmental duplication events were observed among HvPLATZs. All HvPLATZs were core genes present in 20 genotypes of the barley pan-genome. The HvPLATZ5 coding sequences were conserved among 20 barley genotypes, whereas HvPLATZ4/9/10 exhibited synonymous single nucleotide polymorphisms (SNPs); the remaining ones showed nonsynonymous variations. The expression of HvPLATZ2/3/8 was ubiquitous in various tissues, whereas HvPLATZ7 appeared transcriptionally silent; the remaining genes displayed tissue-specific expression. The expression of HvPLATZs was modulated by salt stress, potassium deficiency, and osmotic stress, with response patterns being time-, tissue-, and stress type-dependent. The heterologous expression of HvPLATZ3/5/6/8/9/10/11 in yeast enhanced tolerance to salt and osmotic stress, whereas the expression of HvPLATZ2 compromised tolerance. These results advance our comprehension and facilitate further functional characterization of HvPLATZs.

Genome-wide identification, expression pattern and genetic variation analysis of SWEET gene family in barley reveal the artificial selection of HvSWEET1a during domestication and improvement.

SWEET (Sugars Will Eventually be Exported Transporter) proteins, an essential class of sugar transporters, are involved in vital biological processes of plant growth and development. To date, systematical analysis of SWEET family in barley (Hordeum vulgare) has not been reported. In this study, we genome-wide identified 23 HvSWEET genes in barley, which were further clustered into four clades by phylogenetic tree. The members belonging to the same clade showed relatively similar gene structures and conserved protein motifs. Synteny analysis confirmed the tandem and segmental duplications among HvSWEET genes during evolution. Expression profile analysis demonstrated that the patterns of HvSWEET genes varied and the gene neofunctionalization occurred after duplications. Yeast complementary assay and subcellular localization in tobacco leaves suggested that HvSWEET1a and HvSWEET4, highly expressed in seed aleurone and scutellum during germination, respectively, functioned as plasma membrane hexose sugar transporters. Furthermore, genetic variation detection indicated that HvSWEET1a was under artificial selection pressure during barley domestication and improvement. The obtained results facilitate our comprehensive understanding and further functional investigations of barley HvSWEET gene family, and also provide a potential candidate gene for de novo domestication breeding of barley.

A splicing site change between exon 5 and 6 of the nuclear-encoded chloroplast-localized HvYGL8 gene results in reduced chlorophyll content and plant height in barley.

The chloroplast is an important cellular organelle and metabolic hub, which is not only responsible for plant photosynthesis but is also involved in the de novo biosynthesis of pigments, fatty acids, and hormone metabolisms. Several genes that are responsible for rice leaf color variations have been reported to be directly or indirectly involved in chlorophyll biosynthesis and chloroplast development, whereas a few genes have been functionally confirmed to be responsible for leaf color changes in barley at the molecular level. In this study, we obtained a yellow leaf and dwarf ygl8 mutant from the progeny of Morex (a variety of barley) seeds treated with EMS. We performed bulked-segregant analysis (BSA) and RNA-seq analysis and targeted a UMP kinase encoding gene, YGL8, which generated a splicing site change between exon 5 and 6 of YGL8 due to a G to A single-nucleotide transition in the 5th exon/intron junction in the ygl8 mutant. The splicing site change between exon 5 and 6 of YGL8 had no effects on chloroplast subcellular localization but resulted in an additional loop in the UMP kinase domain, which might disturb the access of the substrates. On one hand, the splicing site change between exon 5 and 6 of YGL8 downregulated the transcriptional expression of chloroplast-encoded genes and chlorophyll-biosynthesis-related genes in a temperature-dependent manner in the ygl8 mutant. On the other hand, the downregulation of bioactive GA-biosynthesis-related GA20ox genes and cell-wall-cellulose-biosynthesis-related CesA genes was also observed in the ygl8 mutant, which led to a reduction in plant height. Our study will facilitate the understanding of the regulation of leaf color and plant height in barley.

Calmodulin and calmodulin-like gene family in barley: Identification, characterization and expression analyses.

Calmodulin (CaM) and calmodulin-like (CML) proteins are Ca2+ relays and play diverse and multiple roles in plant growth, development and stress responses. However, CaM/CML gene family has not been identified in barley (Hordeum vulgare). In the present study, 5 HvCaMs and 80 HvCMLs were identified through a genome-wide analysis. All HvCaM proteins possessed 4 EF-hand motifs, whereas HvCMLs contained 1 to 4 EF-hand motifs. HvCaM2, HvCaM3 and HvCaM5 coded the same polypeptide although they differed in nucleotide sequence, which was identical to the polypeptides coded by OsCaM1-1, OsCaM1-2 and OsCaM1-3. HvCaMs/CMLs were unevenly distributed over barley 7 chromosomes, and could be phylogenetically classified into 8 groups. HvCaMs/CMLs differed in gene structure, cis-acting elements and tissue expression patterns. Segmental and tandem duplication were observed among HvCaMs/CMLs during evolution. HvCML16, HvCML18, HvCML50 and HvCML78 were dispensable genes and the others were core genes in barley pan-genome. In addition, 14 HvCaM/CML genes were selected to examine their responses to salt, osmotic and low potassium stresses by qRT-PCR, and their expression were stress-and time-dependent. These results facilitate our understanding and further functional identification of HvCaMs/CMLs.

Identification of QTL for barley grain size.

BACKGROUND: Barley grain size is one of the key factors determining storage capacity during grain filling. Large, well-filled grains also have a high malt extract potential. Grain size is a complex quantitative trait and can be easily affected by environmental factors thus the identification of genes controlling the trait and the use of molecular markers linked to the genes in breeding program is the most effective way of improving grain size. METHODS: Grain sizes of 188 doubled-haploid (DH) lines derived from the cross of a Japanese malting barley variety (Naso Nijo) and a Chinese feed barley variety (TX9425) were obtained from three different sites in two consecutive years. The average data were used for identifying QTL for grain size. RESULTS: A total of four significant QTL were identified for grain length (GL) and three for grain width (GW). The two major GL QTL are located at similar positions to the QTL for malt extract on 2H and uzu gene on 3H, respectively. However, the GL QTL on 2H is more likely a different one from the malt extract QTL as most of the candidate genes are located outside the fine mapped QTL region for malt extract. The GL QTL on 3H is closely linked with uzu gene but not due to a pleiotropic effect of uzu. The three QTL for grain width on 1H, 2H and 5H, respectively, were located at same position to those for GL.

Identifying Cancer Subtypes Using a Residual Graph Convolution Model on a Sample Similarity Network.

Cancer subtype classification helps us to understand the pathogenesis of cancer and develop new cancer drugs, treatment from which patients would benefit most. Most previous studies detect cancer subtypes by extracting features from individual samples, ignoring their associations with others. We believe that the interactions of cancer samples can help identify cancer subtypes. This work proposes a cancer subtype classification method based on a residual graph convolutional network and a sample similarity network. First, we constructed a sample similarity network regarding cancer gene co-expression patterns. Then, the gene expression profiles of cancer samples as initial features and the sample similarity network were passed into a two-layer graph convolutional network (GCN) model. We introduced the initial features to the GCN model to avoid over-smoothing during the training process. Finally, the classification of cancer subtypes was obtained through a softmax activation function. Our model was applied to breast invasive carcinoma (BRCA), glioblastoma multiforme (GBM) and lung cancer (LUNG) datasets. The accuracy values of our model reached 82.58%, 85.13% and 79.18% for BRCA, GBM and LUNG, respectively, which outperformed the existing methods. The survival analysis of our results proves the significant clinical features of the cancer subtypes identified by our model. Moreover, we can leverage our model to detect the essential genes enriched in gene ontology (GO) terms and the biological pathways related to a cancer subtype.

OsNLA1, a RING-type ubiquitin ligase, maintains phosphate homeostasis in Oryza sativa via degradation of phosphate transporters.

Inorganic phosphate (Pi) transporters (PTs) play vital roles in Pi uptake and translocation in plants. Under Pi sufficient conditions, PTs are degraded to prevent excess Pi accumulation. The mechanisms targeting PTs for degradation are not fully elucidated. In this study, we found that the Oryza sativa (rice) ortholog of Arabidopsis thaliana nitrogen limitation adaptation (NLA), OsNLA1 protein, a RING-type E3 ubiquitin-ligase, was predominantly localized in the plasma membrane, and could interact with rice phosphate transporters OsPT2 and OsPT8. Mutation of the 265th cysteine residue in OsNLA1 that was required for ubiquitination prevented breakdown of OsPT2/PT8, suggesting OsNLA1 targeted OsPT2/PT8 for degradation. Mutation in OsNLA1 (osnla1) led to a significant increase of Pi concentration in leaves in a nitrate-independent manner. Overexpression of OsNLA1 or repression of OsPT2/PT8 restored the high leaf Pi concentration in osnla1 mutants to a level similar to that of wild-type plants. In contrast to what has been observed in Arabidopsis, the transcript abundance of OsNLA1 did not decrease under Pi limited conditions or in OsmiR827 (microRNA827)- or OsPHR2 (PHOSPHATE STARVATION RESPONSE 2)-overexpressing transgenic lines. Moreover, there was no interaction of OsNLA1 and OsPHO2, an E2 ubiquitin-conjugase, suggesting that OsPHO2 was not the partner of OsNLA1 involved in ubiquitin-mediated PT degradation. Our results show that OsNLA1 is involved in maintaining phosphate homeostasis in rice by mediating the degradation of OsPT2 and OsPT8, and OsNLA1 differs from the ortholog in Arabidopsis in several aspects.

Two h-Type Thioredoxins Interact with the E2 Ubiquitin Conjugase PHO2 to Fine-Tune Phosphate Homeostasis in Rice.

Phosphate overaccumulator2 (PHO2) encodes a ubiquitin-conjugating E2 enzyme that is a major negative regulator of the inorganic phosphate (Pi)-starvation response-signaling pathway. A yeast two-hybrid (Y2H) screen in rice (Oryza sativa; Os) using OsPHO2 as bait revealed an interaction between OsPHO2 and two h-type thioredoxins, OsTrxh1 and OsTrxh4. These interactions were confirmed in vivo using bimolecular fluorescence complementation (BiFC) of OsPHO2 and OsTrxh1/h4 in rice protoplasts and by in vitro pull-down assays with 6His-tagged OsTrxh1/h4 and GST-tagged OsPHO2. Y2H assays revealed that amino acid Cys-445 of OsPHO2 and an N-terminal Cys in the "WCGPC" motif of Trxhs were required for the interaction. Split-ubiquitin Y2H analyses and BiFC assays in rice protoplasts confirmed the interaction of OsPHO2 with PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (OsPHF1), and PHOSPHATE1;2 (OsPHO1;2) in the endoplasmic reticulum and Golgi membrane system, where OsPHO2 mediates the degradation of OsPHF1 in both tobacco (Nicotiana benthamiana) leaves and rice seedlings. Characterization of rice pho2 complemented lines, transformed with an endogenous genomic OsPHO2 or OsPHO2C445S (a constitutively reduced form) fragment, indicated that OsPHO2C445S restored Pi concentration in rice to statistically significant lower levels compared to native OsPHO2 Moreover, the suppression of OsTrxh1 (knockdown and knockout) resulted in slightly higher Pi concentration than that of wild-type Nipponbare in leaves. These results demonstrate that OsPHO2 is under redox control by thioredoxins, which fine-tune its activity and link Pi homeostasis with redox balance in rice.

Mutation of OsGIGANTEA Leads to Enhanced Tolerance to Polyethylene Glycol-Generated Osmotic Stress in Rice.

Water deficit is one of the most important environmental stresses limiting plant growth and crop yield. While the identification of many key factors involved in the plant water deficit response has greatly increased our knowledge about the regulation system, the mechanisms underlying dehydration tolerance in plants are still not well understood. In our current study, we investigated the roles of the key flowering time regulator, OsGIGANTEA (OsGI), in the osmotic stress tolerance in rice. Results showed that mutation of OsGI conferred tolerance to osmotic stress generated by polyethylene glycol (PEG), increased proline and sucrose contents, and accelerated stomata movement. In addition, qRT-PCR and microarray analysis revealed that the transcript abundance of some osmotic stress response genes, such as OsDREB1E, OsAP37, OsAP59, OsLIP9, OsLEA3, OsRAB16A, and OsSalT, was significantly higher in osgi than in WT plants, suggesting that OsGI might be a negative regulator in the osmotic stress response in rice.

Rice SPX-Major Facility Superfamily3, a Vacuolar Phosphate Efflux Transporter, Is Involved in Maintaining Phosphate Homeostasis in Rice.

To maintain a stable cytosol phosphate (Pi) concentration, plant cells store Pi in their vacuoles. When the Pi concentration in the cytosol decreases, Pi is exported from the vacuole into the cytosol. This export is mediated by Pi transporters on the tonoplast. In this study, we demonstrate that SYG1, PHO81, and XPR1 (SPX)-Major Facility Superfamily (MFS) proteins have a similar structure with yeast (Saccharomyces cerevisiae) low-affinity Pi transporters Phosphatase87 (PHO87), PHO90, and PHO91. OsSPX-MFS1, OsSPX-MFS2, and OsSPX-MFS3 all localized on the tonoplast of rice (Oryza sativa) protoplasts, even in the absence of the SPX domain. At high external Pi concentration, OsSPX-MFS3 could partially complement the yeast mutant strain EY917 under pH 5.5, which lacks all five Pi transporters present in yeast. In oocytes, OsSPX-MFS3 was shown to facilitate Pi influx or efflux depending on the external pH and Pi concentrations. In contrast to tonoplast localization in plants cells, OsSPX-MFS3 was localized to the plasma membrane when expressed in both yeast and oocytes. Overexpression of OsSPX-MFS3 results in decreased Pi concentration in the vacuole of rice tissues. We conclude that OsSPX-MFS3 is a low-affinity Pi transporter that mediates Pi efflux from the vacuole into cytosol and is coupled to proton movement.