BmSuc1, a novel animal-type β-fructofuranosidase (β-FFase, EC 3.2.1.26) encoding gene, was cloned and identified for the first time in the silkworm, Bombyx mori. BmSuc1 was specifically and highly expressed in the midgut and silk gland of Bombyx mori. Until now, the function of BmSuc1 in the silk gland was unclear. In this study, it was found that the expression changes of BmSuc1 in the fifth instar silk gland were consistent with the growth rate of the silk gland. Next, with the aid of the CRISPR/Cas9 system, the BmSuc1 locus was genetically mutated, and homozygous mutant silkworm strains with truncated β-FFase (BmSUC1) proteins were established. BmSuc1 mutant larvae exhibited stunted growth and decreased body weight. Interestingly, the molecular weight of part of Sericin1 (Ser1) in the silk gland of the mutant silkworms was reduced. The knockout of BmSuc1 reduced the sericin content in the silkworm cocoon shell, and the mechanical properties of the mutant line silk fibers were also negatively affected. These results reveal that BmSUC1 is involved in the synthesis of Ser1 protein in silk glands and helps to maintain the homeostasis of silk protein content in silk fibers and the mechanical properties of silk fibers, laying a foundation for the study of BmSUC1 regulation of silk protein synthesis in silk glands.

β-fructofuranosidase (β-FFase) belongs to the glycosyl-hydrolase family 32 (GH32), which can catalyze both the release of β-fructose from β-d-fructofuranoside substrates to hydrolyze sucrose and the synthesis of short-chain fructooligosaccharide (FOS). BmSuc1 has been cloned and identified from the silkworm Bombyx mori as a first animal type of β-FFase encoding gene. It was hypothesized that BmSUC1 plays an important role in the silkworm-mulberry adaptation system. However, there is little information about the enzymatic core sites of BmSUC1. In this study, we mutated three amino acid residues (D63, D181, and E234) that represent important conserved motifs for β-FFase activity in GH32 to alanine respectively by using site-directed mutagenesis. Recombinant proteins of three mutants and wild type BmSUC1 were obtained by using a Bac-to-Bac/BmNPV expression system and BmN cells. Enzymatic activity, kinetic properties, and substrate specificity of the four proteins were analyzed. High Performance Liquid Chromatography (HPLC) was used to compare the hydrolyzing and transfructosylating activities between D181A and wtBmSUC1. Our results revealed that the D63A and E234A mutations lost activity, suggesting that D63 and E234 are key amino acid residues for BmSUC1 to function as an enzyme. The D181A mutation significantly enhanced both hydrolyzing and transfructosylating activities of BmSUC1, indicating that D181 may not be directly involved in catalyzation. The results provide insight into the chemical catalyzation mechanism of BmSUC1 in B. mori. Up-regulated transfructosylating activity of BmSUC1 could provide new ideas for using B. mori β-FFase to produce functional FOS.

Increasing rice yield has always been one of the primary objectives of rice breeding. However, panicle degeneration, a complex phenomenon regulated by many genetic and environmental factors, often occurs in rice-growing regions and severely curbs rice yield. In this study, we obtained a new apical panicle degeneration mutant named ym48, which induces a marked degeneration rate and diminishes the final grain yield. Cellular and physiological analyses revealed that the apical panicle in ym48 undergoes programmed cell death, accompanied by excessive accumulations of peroxides. Following, the panicle degeneration gene OsCAX1a was identified, which was involved in Ca2+ transport in the ym48 mutant. In OsCAX1a, a relative conserved T to A substitution was noted at the 64th amino acid, which disrupted Ca2+ transport. Hydroponics assays and Ca2+ quantification confirmed that Ca2+ transport and distribution to apical tissues were restricted and over-accumulated in mutant sheath. Ca2+ transport between cytoplasm and vacuole was affected, and the reduced content of Ca2+ in vacuole and cell wall and the decreased of Ca2+ absorption were appeared in ym48 mutant. RNA-Seq data indicated that the abnormal CBL (calcineurin b-like proteins) pathway mediated by deficient Ca2+ might occur in mutant, resulting in the burst of ROS and programmed cell death in panicles. Our results explained the key role of OsCAX1a in Ca2+ transport and distribution and laid a foundation to further explore the genetic and molecular mechanisms of panicle degeneration and the efficiency of Ca2+ fertilization in rice.

Increasing the yield of rice has always been one of the primary purposes of rice breeding. However, panicle degeneration, a complex characteristic regulated by many genes and commonly encountered in rice production, seriously reduces yield. In this study, we obtained a new apical panicle degeneration mutant named ym48 , which induces a marked degeneration rate and reduces grain yield in rice. Cellular and physiological analyses revealed that the apical panicle in ym48 undergoes programmed cell death, accompanied by the accumulation of higher levels of peroxide. Next, the OsCAX1a gene, which is responsible for Ca ²⁺ transportation, was identified, and a highly conserved A to G substitution was noted at the 190 ^th nucleotide in ym48 , which seriously damaged Ca ²⁺ transport. Through hydroponics assays and Ca ²⁺ quantification, we found that Ca ²⁺ transportation and distribution from the underground part to apical tissues was blocked and overaccumulated in mutant stems. In general, this Ca ²⁺ deficiency mediated by OsCAX1a induced peroxide accumulation and resulted in programmed cell death in the apical panicle of the ym48 mutant. Our results demonstrate the essential role of OsCAX1a in Ca ²⁺ transportation and distribution, laying a foundation to further explore the genetic and molecular mechanisms of panicle development and differentiation in rice.