Basic helix-loop-helix (bHLH) transcription factors are involved in many abiotic stress responses as well as flavonol and anthocyanin biosynthesis. In grapes (Vitis vinifera L.), flavonols including anthocyanins and condensed tannins are most abundant in the skins of the berries. Flavonols are important phytochemicals for viticulture and enology, but grape bHLH genes have rarely been examined. We identified 94 grape bHLH genes in a genome-wide analysis and performed Nr and GO function analyses for these genes. Phylogenetic analyses placed the genes into 15 clades, with some remaining orphans. 41 duplicate gene pairs were found in the grape bHLH gene family, and all of these duplicate gene pairs underwent purifying selection. Nine triplicate gene groups were found in the grape bHLH gene family and all of these triplicate gene groups underwent purifying selection. Twenty-two grape bHLH genes could be induced by PEG treatment and 17 grape bHLH genes could be induced by cold stress treatment including a homologous form of MYC2, VvbHLH007. Based on the GO or Nr function annotations, we found three other genes that are potentially related to anthocyanin or flavonol biosynthesis: VvbHLH003, VvbHLH007, and VvbHLH010. We also performed a cis-acting regulatory element analysis on some genes involved in flavonoid or anthocyanin biosynthesis and our results showed that most of these gene promoters contained G-box or E-box elements that could be recognized by bHLH family members.

Plant long non-coding RNA (lncRNA) undergoes dynamic regulation and acts in developmental and stress regulation. In this study, we surveyed the expression dynamics of lncRNAs in grapevine (Vitis vinifera L.) under cold stress using high-throughput sequencing. Two-hundred and three known lncRNAs were significantly up-regulated and 144 known lncRNAs were significantly down-regulated in cold-treated grapevine. In addition, 2 088 novel lncRNA transcripts were identified in this study, with 284 novel lncRNAs significantly up-regulated and 182 novel lncRNAs significantly down-regulated in cold-treated grapevine. Two-hundred and forty-two differentially expressed grapevine lncRNAs were predicted to target 326 protein-coding genes in a cis-regulatory relationship. Many differentially expressed grapevine lncRNAs targeted stress response-related genes, such as CBF4 transcription factor genes, late embryogenesis abundant protein genes, peroxisome biogenesis protein genes, and WRKY transcription factor genes. Sixty-two differentially expressed grapevine lncRNAs were predicted to target 100 protein-coding genes in a trans-regulatory relationship. The expression of overall target genes in both cis and trans-regulatory relationships were positively related to the expression of lncRNAs in grapevines under cold stress. We identified 31 known lncRNAs as 34 grapevine micro RNA (miRNA) precursors and some miRNAs may be derived from multiple lncRNAs. We found 212 lncRNAs acting as targets of miRNAs in grapevines, involving 150 miRNAs; additionally, 120 grapevine genes were predicted as targets of grapevine miRNAs and lncRNAs. We found one gene cluster that was up-regulated and showed the same expression trend. In this cluster, many genes may be involved in abiotic stress response such as WRKY, Hsf, and NAC transcription factor genes.

Foxtail millet is very a drought-tolerant crop. Basic helix–loop–helix (bHLH) transcription factors are involved in many drought-stress responses, but foxtail millet bHLH genes have been scarcely examined. We identified 149 foxtail millet bHLH genes in a genome-wide analysis and performed Swiss-Prot, GO, and KEGG pathway analyses for these genes. Phylogenetic analyses placed the genes into 25 clades, with some remaining orphans. We identified homologs based on gene trees and Swiss-Prot annotation. We also inferred that some homologs underwent positive selection in foxtail millet ancestors, and selected motifs differed among homologs. Expression of eight foxtail millet bHLH genes varied with drought stress. One of these genes was localized to a QTL that contributes to drought tolerance in foxtail millet. We also perform a cis-acting regulatory element analysis on foxtail millet bHLH genes and some drought-induced genes. Foxtail millet bHLH genes were inferred to have a possible key role in drought tolerance. This study clarifies both the function of foxtail millet bHLH genes and drought tolerance in foxtail millet.

Chinese wild grapevine (Vitis amurensis) has remarkable cold stress tolerance, exceeding that of the common cultivated grapevine (Vitis vinifera L.). Here, we surveyed the expression dynamics of microRNAs (miRNAs) across Chinese wild grapevine (cv. Beibinghong) and cultivated grapevine (cv. Cabernet Sauvignon) under cold stress using high-throughput sequencing. We identified 186 known miRNAs in cultivated grape and 427 known miRNAs in Beibinghong. Of the identified miRNAs, 59 are conserved miRNAs orthologous in Cabernet Sauvignon and Beibinghong. In addition, 105 and 129 novel miRNAs were identified in Cabernet Sauvignon and Beibinghong, respectively. The expression of some miRNAs was related to cold stress both in Cabernet Sauvignon and Beibinghong. Many cold-related miRNAs in Cabernet Sauvignon and Beibinghong were predicted to target stress response-related genes such as MYB, WRKY, bHLH transcription factor genes, and heat shock protein genes. However, the expression tendency under cold treatment of many of these miRNAs was different between Cabernet Sauvignon and Beibinghong. Different modes of expression of cultivated and Chinese wild grape miRNAs were indicated in key pathways under cold stress by degradome, target prediction, GO, and KEGG analyses. Our study indicated three likely reasons that led to the different cold stress tolerance levels of Cabernet Sauvignon and Beibinghong. Specifically, there may be (1) differential expression of orthologous miRNAs between cultivated grapevine and Chinese wild grape; (2) species-specific miRNAs or target genes; or (3) different regulatory models of miRNAs in cultivated and Chinese wild grape in some key pathways.

CLE genes play various biological roles in plant growth and development, as well as in responses to environmental stimuli. In the present study, we identified nine CLE genes in the grape genome using an effective identification method. We analyzed the expression profiles of grape CLE genes in different tissues and under environmental different stimuli. VvCLE3 was expressed in shoot apical meristem (SAM) enriched regions, and VvCLE6 was expressed in shoot tissue without SAM. When grapes were infected with bois noir, VvCLE2 was up-regulated. Under ABA treatment, VvCLE3 was down-regulated. VvCLE6 was up-regulated under high temperature stress. We found that VvCLE6 and VvCLE1 were highly expressed in root tissue. In addition, we compared the characteristics of CLEs from grape and other plant species. The CLE family in Sphagnum fallax underwent positive selection, while the CLE family in grape underwent purifying selection. The frequency of optimal codons and codon adaptation index of rice and grape CLE family members were positively correlated with GC content at the third site of synonymous codons, indicating that the dominant evolutionary pressure acting on rice and grape CLE genes was mutation pressure. We also found that closely related species had higher levels of similarity in relative synonymous codon usage in CLE genes. The rice CLE family was biased toward C and G nucleotides at third codon positions. Gene duplication and loss events were also found in grape CLE genes. These results demonstrate an effective identification method for CLE motifs and increase the understanding of grape CLEs. Future research on CLE genes may have applications for grape breeding and cultivation to better understand root and nodulation development.

Molybdenum (Mo) is a component of the Mo cofactor (Moco) of nitrate reductase (NR) and is therefore essential for nitrate metabolism. However, little is known about Mo deficiency phenotypes or about how physiological and molecular mechanisms of Mo uptake and transport influence nitrate uptake and utilization in strawberry. Here, we used physiological and cytological techniques to identify Mo deficiency phenotypes in strawberry. Seedlings cultured with MoO₄^2- grew well and exhibited normal microstructure and ultrastructure of leaves and roots. By contrast, seedlings cultivated under Mo-deficient conditions showed yellow leaf blades and ultrastructural changes such as irregular chloroplasts and unclear membrane structures that were similar to the symptoms of nitrogen deficiency. We cloned and analyzed a putative molybdate transporter, FaMOT1, which may encode a molybdate transporter involved in the uptake and translocation of molybdate. Interestingly, the addition of the molybdate analog tungstate led to lower tissue Mo concentrations, reduced the translocation of Mo from roots to shoots, and increased the plants’ sensitivity to Mo deficiency. Seedlings cultivated with MoO₄^2- altered expression of genes in Moco biosynthesis. As expected, NR activity was higher under sufficient MoO₄^2- levels. Furthermore, seedlings grown on Mo-deficient medium exhibited decreased ¹⁵NO₃^- translocation and lower ¹⁵NO₃^- use efficiency. These findings represent an important step towards understanding how molybdate transport, concentration, and deficiency symptoms influence nitrate uptake and utilization in strawberry.