Genome-wide identification, evolution, and role of SPL gene family in beet (Beta vulgaris L.) under cold stress.

BACKGROUND: SPL transcription factors play vital roles in regulating plant growth, development, and abiotic stress responses. Sugar beet (Beta vulgaris L.), one of the world's main sugar-producing crops, is a major source of edible and industrial sugars for humans. Although the SPL gene family has been extensively identified in other species, no reports on the SPL gene family in sugar beet are available. RESULTS: Eight BvSPL genes were identified at the whole-genome level and were renamed based on their positions on the chromosome. The gene structure, SBP domain sequences, and phylogenetic relationship with Arabidopsis were analyzed for the sugar beet SPL gene family. The eight BvSPL genes were divided into six groups (II, IV, V, VI, VII, and VIII). Of the BvSPL genes, no tandem duplication events were found, but one pair of segmental duplications was present. Multiple cis-regulatory elements related to growth and development were identified in the 2000-bp region upstream of the BvSPL gene start codon (ATG). Using quantitative real-time polymerase chain reaction (qRT-PCR), the expression profiles of the eight BvSPL genes were examined under eight types of abiotic stress and during the maturation stage. BvSPL transcription factors played a vital role in abiotic stress, with BvSPL3 and BvSPL6 being particularly noteworthy. CONCLUSION: Eight sugar beet SPL genes were identified at the whole-genome level. Phylogenetic trees, gene structures, gene duplication events, and expression profiles were investigated. The qRT-PCR analysis indicated that BvSPLs play a substantial role in the growth and development of sugar beet, potentially participating in the regulation of root expansion and sugar accumulation.

FtbZIP12 Positively Regulates Responses to Osmotic Stress in Tartary Buckwheat.

ABFs play a key role in regulating plant osmotic stress. However, in Tartary buckwheat, data on the role of ABF genes in osmotic stress remain limited and its associated mechanism in osmoregulation remain nebulous. Herein, a novel ABF family in Tartary buckwheat, FtbZIP12, was cloned and characterized. FtbZIP12 is a transcriptional activator located in the nucleus; its expression is induced by NaCl, mannitol, and abscisic acid (ABA). Atopic expression of FtbZIP12 in Arabidopsis promoted seed germination, reduced damage to primary roots, and improved the tolerance of seedlings to osmotic stress. The quantitative realtime polymerase chain reaction (RT-qPCR) results showed that the expressions of the typical genes related to stress, the SOS pathway, and the proline synthesis pathway in Arabidopsis were significantly (p < 0.05) upregulated under osmotic stress. FtbZIP12 improved the osmotic pressure resistance by reducing the damage caused by reactive oxygen species to plants and maintained plant homeostasis by upregulating the expression of genes related to stress, osmotic regulation, and ion homeostasis. This study identified a key candidate gene for understanding the mechanism underlying osmotic-stress-regulated function in Tartary buckwheat, thereby providing a theoretical basis for improving its yield and quality.

Genome-Wide Identification, Evolution, and Expression Pattern Analysis of the GATA Gene Family in Tartary Buckwheat (Fagopyrum tataricum).

GATA is a transcription factor that exerts a vital function in plant growth and development, physiological metabolism, and environmental responses. However, the GATA gene family has rarely been studied in Tartary buckwheat since the completion of its genome. This study used bioinformatics methods to identify GATA genes of Tartary buckwheat and to analyze their subfamily classification, structural composition, and developmental evolution, as well as to discuss the expression patterns of FtGATA genes in different subfamilies. The twenty-eight identified FtGATA genes in the Tartary buckwheat genome were divided into four subfamilies and distributed on eight chromosomes. One pair of tandem repeat genes and eight pairs of fragments were found in chromosome mapping. Spatiotemporal expression patterns of eight FtGATA genes in different subfamilies indicated that the FtGATA gene family has regulatory roles in tissue specificity, fruit development, abiotic stress, and hormonal responses. This study creates a theoretical and scientific foundation for further research on the evolutionary relationship and biological function of FtGATA.

Physiological and Biochemical Regulation Mechanism of Exogenous Hydrogen Peroxide in Alleviating NaCl Stress Toxicity in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn).

We aimed to elucidate the physiological and biochemical mechanism by which exogenous hydrogen peroxide (H2O2) alleviates salt stress toxicity in Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn). Tartary buckwheat "Chuanqiao-2" under 150 mmol·L-1 salt (NaCl) stress was treated with 5 or 10 mmol·L-1 H2O2, and seedling growth, physiology and biochemistry, and related gene expression were studied. Treatment with 5 mmol·L-1 H2O2 significantly increased plant height (PH), fresh and dry weights of shoots (SFWs/SDWs) and roots (RFWs/RDWs), leaf length (LL) and area (LA), and relative water content (LRWC); increased chlorophyll a (Chl a) and b (Chl b) contents; improved fluorescence parameters; enhanced antioxidant enzyme activity and content; and reduced malondialdehyde (MDA) content. Expressions of all stress-related and enzyme-related genes were up-regulated. The F3'H gene (flavonoid synthesis pathway) exhibited similar up-regulation under 10 mmol·L-1 H2O2 treatment. Correlation and principal component analyses showed that 5 mmol·L-1 H2O2 could significantly alleviate the toxic effect of salt stress on Tartary buckwheat. Our results show that exogenous 5 mmol·L-1 H2O2 can alleviate the inhibitory or toxic effects of 150 mmol·L-1 NaCl stress on Tartary buckwheat by promoting growth, enhancing photosynthesis, improving enzymatic reactions, reducing membrane lipid peroxidation, and inducing the expression of related genes.

Comparison of buckwheat genomes reveals the genetic basis of metabolomic divergence and ecotype differentiation.

Golden buckwheat (Fagopyrum dibotrys or Fagopyrum cymosum) and Tartary buckwheat (Fagopyrum tataricum) belong to the Polygonaceae and the Fagopyrum genus is rich in flavonoids. Golden buckwheat is a wild relative of Tartary buckwheat, yet golden buckwheat is a traditional Chinese herbal medicine and Tartary buckwheat is a food crop. The genetic basis of adaptive divergence between these two buckwheats is poorly understood. Here, we assembled a high-quality chromosome-level genome of golden buckwheat and found a one-to-one syntenic relationship with the chromosomes of Tartary buckwheat. Two large inversions were identified that differentiate golden buckwheat and Tartary buckwheat. Metabolomic and genetic comparisons of golden buckwheat and Tartary buckwheat indicate an amplified copy number of FdCHI, FdF3H, FdDFR, and FdLAR gene families in golden buckwheat, and a parallel increase in medicinal flavonoid content. Resequencing of 34 wild golden buckwheat accessions across the two morphologically distinct ecotypes identified candidate genes, including FdMYB44 and FdCRF4, putatively involved in flavonoid accumulation and differentiation of plant architecture, respectively. Our comparative genomic study provides abundant genomic resources of genomic divergent variation to improve buckwheat with excellent nutritional and medicinal value.

Purification and properties of the chymotrypsin inhibitor from wild emmer wheat (Triticum dicoccoides) of Israel and its toxic effect on beet armyworm, Spodoptera exigua.

A novel chymotrypsin inhibitor, which detected in the seed of wild emmer wheat (Triticum dicoccoides), was purified by ion-exchange chromatography, affinity chromatography and Ultracentrifugation. On the basis of its specificity, this inhibitor was named WeCI (wild emmer chymotrypsin inhibitor). SDS-PAGE analysis displayed that the purified WeCI is a single chain polypeptide with a molecular weight of approximately 13kDa. The inhibition constants (Ki) for amylase and bovine pancreatic chymotrypsin were 1.12×10-9M and 2.41×10-9M, respectively. Automated sequencing and mass spectrometry analyses revealed that WeCI is a neutral monomeric protein consisting of 119 residues. In vitro, WeCI strongly suppressed bovine pancreatic chymotrypsin as well as chymotrypsin-like activities separated from the midgut of the beet armyworm Spodoptera exigua. No inhibitory activities were found against bovine pancreatic trypsin, bacterial subtilisin, or porcine pancreatic elastase. The primary structure of WeCI was markedly similar (46-95%) to those of several proteins belonging to the wheat crop chymotrypsin/α-amylase inhibitor superfamily and displayed the typical sequence motif of the α-amylase inhibitor-seed storage protein group. WeCI significantly inhibited the growth and development of Spodoptera exigua, dependent on inhibitor concentration. WeCI significantly increased the mortality rate of Spodoptera exigua and caused a significant decrease in its fertility.

Expression and purification of the trypsin inhibitor from tartary buckwheat in Pichia pastoris and its novel toxic effect on Mamestra brassicae larvae.

The gene of the trypsin inhibitor of tartary buckwheat (Fagopyrum tataricum) was successfully cloned, expressed in Pichia pastoris and tested for regulatory effects on insect growth. The three significant factors were optimized by single-factor experiments and central composite design in response surface methodology. Proteins were efficiently expressed at levels of 489.6-527.4 U/mg in shaken flasks. The trypsin inhibitor from tartary buckwheat (FtTI) was purified by affinity chromatography and centrifugal ultrafiltration. The purified FtTI efficiently inhibited trypsin protease activity by competitive inhibition with a Ki value 1.5 nM. The molecular mass of the purified protein was approximately 13.8 kDa. FtTI had a higher toxic killing effect on Mamestra brassicae larvae. The median lethal concentration for the larvae was 15 µg/mL.

An antifungal peptide from Fagopyrum tataricum seeds.

A major trypsin inhibitor was isolated and characterized from the seeds of the tartary buckwheat (Fagopyrum tataricum) (FtTI) by ammonium sulfate precipitation, ion exchange chromatography and centrifugal ultrafiltration. SDS-PAGE analysis under reducing condition showed that FtTI is a single polypeptide chain with a molecular mass of approximately 14kDa. The complete amino acid sequence of FtTI was established by automatic Edman degradation and mass spectrometry. It was found that the trypsin inhibitor molecule consists of 86 amino acid residues containing two disulfide bonds which connect Cys(8) to Cys(65) and Cys(49) to Cys(58). The active site of the inhibitor was found to contain an Asp(66)-Arg(67) bond. MALDI-TOF analysis showed that FtTI has two isoforms (Mr: 11.487 and 13.838kDa). Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) of 1.6nM. Analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor I family. What is more, FtTI exhibited strong inhibitory activity against phytopathogenic fungi.