Recombinant Oryza sativa subsp. japonica Beta-galactosidase 14 (Os10g0340600, LOC_Os10g19960), partial

What is the phylogenetic classification of OsBgal14?

OsBgal14 belongs to glycosyl hydrolase family 35 (GH35), which comprises enzymes with β-galactosidase activity. Within the rice β-galactosidase family, OsBgal14 is classified in phylogenetic group b, which contains four rice Bgals (OsBgal5, OsBgal12, OsBgal14, and OsBgal15) with high amino acid sequence similarity. These isozymes have nearly identical protein lengths and gene structures, suggesting they evolved from a common ancestor through gene duplication events. The rice genome contains a total of 15 β-galactosidase genes, similar to the 17 found in Arabidopsis, indicating conservation of this gene family across plant species .

What is the predicted protein structure of OsBgal14?

OsBgal14, like other plant β-galactosidases, likely contains a catalytic domain typical of GH35 enzymes and a C-terminal galactose-binding lectin-like domain. This C-terminal domain is a common feature among plant-type β-galactosidases and is thought to play a role in substrate recognition and binding. The protein is predicted to contain a secretory signal sequence, suggesting it functions in the secretory pathway or external cellular environment. Based on structural analyses of related β-galactosidases, the catalytic domain likely adopts a TIM barrel fold with conserved active site residues responsible for hydrolytic activity .

What is the tissue-specific expression pattern of OsBgal14?

OsBgal14 appears to have a reproductive tissue-specific expression pattern. According to transcript expression analysis, OsBgal14 belongs to a distinct lineage of rice β-galactosidases (group b) that are expressed nearly exclusively in reproductive tissues. This expression pattern is similar to certain Arabidopsis β-galactosidases that are expressed most highly in flowers and pollen. This suggests that OsBgal14 may play specialized roles during reproductive development in rice, potentially involving cell wall modification or remodeling during these developmental processes .

How does OsBgal14 compare to other rice β-galactosidases?

OsBgal14 shows high sequence similarity to three other rice β-galactosidases: OsBgal5, OsBgal12, and OsBgal15. These four enzymes form a distinct phylogenetic group (group b) and likely have similar functions based on their sequence similarity and expression patterns. In contrast, other rice β-galactosidases fall into different phylogenetic groups with distinct expression patterns and putative functions. For example, OsBgal6 is predominantly expressed in young leaves and roots, while OsBgal1, OsBgal2, and OsBgal13 are expressed in multiple tissues including germinating seeds and developing grains .

What are the predicted substrates for OsBgal14?

While specific substrate information for OsBgal14 is not directly reported, predictions can be made based on related β-galactosidases. Plant β-galactosidases typically hydrolyze β-galactosidic linkages in various substrates including cell wall polysaccharides, glycoproteins, and glycolipids. Based on its reproductive tissue-specific expression, OsBgal14 may act on specialized galactose-containing substrates present in these tissues. Other characterized plant β-galactosidases have been shown to hydrolyze β-(1→4)-galactan in pectin (Class I enzymes) or β-(1→3)- and β-(1→6)-galactosyl linkages in arabinogalactan proteins (Class II enzymes). Further biochemical analysis would be needed to determine the specific substrate preference of OsBgal14 .

What regulatory mechanisms control OsBgal14 expression?

The reproductive tissue-specific expression of OsBgal14 suggests the presence of tissue-specific regulatory elements in its promoter region. While the specific transcription factors controlling OsBgal14 expression are not reported, its co-expression with other reproductive tissue-specific genes might provide clues about shared regulatory mechanisms. Alternative splicing, which has been observed in other rice β-galactosidase genes like OsBgal6 and OsBgal11, could potentially affect OsBgal14 expression as well. In OsBgal6, alternative splicing in panicle tissues prevents the production of full-length protein, serving as a post-transcriptional regulatory mechanism. Similar mechanisms might exist for fine-tuning OsBgal14 expression during reproductive development .

How might OsBgal14 function differ from constitutively expressed β-galactosidases?

OsBgal14's reproductive tissue-specific expression distinguishes it from constitutively expressed β-galactosidases like OsBgal1 and OsBgal2, which are present in multiple tissues. This specialized expression pattern suggests OsBgal14 likely fulfills specific functions during reproductive development rather than participating in general cell wall maintenance. The enzyme may be involved in processes such as pollen tube growth, anther dehiscence, or flower development, all of which involve dynamic cell wall modifications. The restricted expression pattern might also reflect adaptation to specific substrates or cellular environments present only in reproductive tissues .

What evolutionary history does OsBgal14 share with other plant β-galactosidases?

Phylogenetic analysis indicates that OsBgal14 belongs to a nearly plant-specific subfamily of β-galactosidases, most of which contain a C-terminal lectin-like domain. This subfamily evolved separately from another type of β-galactosidase represented by OsBgal9, which clusters with animal β-galactosidases. The presence of both types of β-galactosidases in the moss Physcomitrella patens suggests they diverged early in plant evolution, before the emergence of vascular plants. Within the plant-specific subfamily, OsBgal14 and its close relatives (OsBgal5, OsBgal12, and OsBgal15) likely arose through gene duplication events specific to the rice lineage or common to grass species .

Could alternative splicing affect OsBgal14 function?

Alternative splicing has been observed in several rice β-galactosidase genes, notably OsBgal6 and OsBgal11. In OsBgal6, alternative splicing in panicle tissue produces a transcript with a premature stop codon, resulting in a truncated, non-functional protein. This mechanism appears to prevent OsBgal6 expression in tissues where its function may be unnecessary or detrimental. Given that OsBgal14 belongs to the same gene family, it might utilize similar post-transcriptional regulatory mechanisms. Investigation of potential alternative splicing events in OsBgal14 could reveal important insights into its regulation during reproductive development .

What is the subcellular localization of OsBgal14 and how does it relate to function?

OsBgal14, like all 15 rice β-galactosidase proteins, is predicted to contain a secretory signal sequence, suggesting it functions in the secretory pathway or extracellular space. This localization is consistent with its potential role in cell wall modification, as substrates like pectin, xyloglucan, and arabinogalactan proteins are components of the cell wall or middle lamella. The specific subcellular compartment where OsBgal14 accumulates (e.g., cell wall, apoplast, or vacuole) would influence which substrates it encounters and thus its biological function. Determining the precise subcellular localization through techniques like immunolocalization or fluorescent protein fusions would provide valuable insights into OsBgal14 function .

What cloning strategies are effective for recombinant OsBgal14 expression?

For recombinant expression of OsBgal14, researchers should first isolate the full-length coding sequence from reproductive tissue cDNA, where the gene is predominantly expressed. Based on successful approaches with other β-galactosidases, the gene can be amplified using specific primers designed from the annotated sequence (Os10g0340600, LOC_Os10g19960). After PCR amplification, the gene can be cloned into an appropriate expression vector, such as pET28a(+), which provides an N-terminal His-tag for purification. When designing the expression construct, researchers should consider whether to include or exclude the native signal peptide, depending on the desired localization of the recombinant protein. For expression in E. coli, removing the signal peptide is typically recommended to prevent potential issues with protein folding and solubility .

How can OsBgal14 enzyme activity be measured effectively?

β-Galactosidase activity can be measured using synthetic substrates like o-nitrophenyl-β-D-galactopyranoside (ONPG). In a standard assay, the enzyme sample is mixed with ONPG solution and incubated at the optimal temperature (typically determined empirically, often around 40°C for β-galactosidases). The reaction is stopped by adding Na₂CO₃, and the released o-nitrophenol (ONP) is measured spectrophotometrically at 420 nm. One unit of β-galactosidase activity is defined as the amount of enzyme required to release 1 μmol of ONP per minute under the assay conditions. For more specific analysis of substrate preferences, researchers can use various natural substrates such as lactose, raffinose, or plant cell wall-derived oligosaccharides, with product formation monitored by techniques like thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC) .

What expression systems are optimal for recombinant OsBgal14 production?

While E. coli is commonly used for recombinant protein expression due to its simplicity and high yield, plant proteins often require eukaryotic expression systems for proper folding and post-translational modifications. For OsBgal14, researchers might consider:

• E. coli: Suitable for initial characterization but may result in inclusion bodies requiring refolding.

• Yeast systems (Pichia pastoris or Saccharomyces cerevisiae): Provide eukaryotic processing while maintaining relatively high yields.

• Insect cell lines: Offer advanced eukaryotic processing capabilities.

• Plant expression systems: Provide the most native environment for protein folding but typically with lower yields.

The choice depends on the research goals - E. coli expression might be sufficient for preliminary activity assays, while studies of glycosylation patterns or complex substrate interactions might require plant-based expression systems .

How can researchers investigate OsBgal14 substrate specificity?

To determine OsBgal14 substrate specificity, researchers should test the purified recombinant enzyme against various potential substrates:

• Synthetic substrates: ONPG provides a convenient colorimetric assay for initial activity confirmation.

• Natural substrates: Testing activity on lactose, raffinose, and plant-specific oligosaccharides.

• Cell wall-derived polysaccharides: Including β-(1→4)-galactan from pectin, β-(1→3)- and β-(1→6)-linked galacto-oligosaccharides from arabinogalactan proteins.

Products can be analyzed using:

• TLC for qualitative analysis of hydrolysis products

• HPLC for quantitative determination of product formation rates

• Mass spectrometry for detailed structural analysis of complex oligosaccharide hydrolysis products

Kinetic parameters (Km, Vmax) should be determined for substrates showing significant hydrolysis to characterize the enzyme's catalytic efficiency .

What approaches can reveal the physiological role of OsBgal14 in planta?

To understand the physiological function of OsBgal14 in rice plants, researchers can employ several complementary approaches:

• Gene expression analysis: Detailed RT-PCR or RNA-Seq analysis across reproductive tissues and developmental stages to precisely define temporal and spatial expression patterns.

• Reverse genetics:

  • RNAi or CRISPR-Cas9 to generate OsBgal14 knockdown or knockout lines

  • Phenotypic analysis focusing on reproductive development, pollen viability, and seed setting

• Overexpression studies: Constitutive or tissue-specific overexpression to observe gain-of-function phenotypes.

• Immunolocalization: Developing specific antibodies to determine the precise tissue and subcellular localization of the native protein.

• Metabolite analysis: Comparing cell wall composition between wild-type and mutant plants to identify accumulated substrates in the absence of OsBgal14 activity.

These approaches together can provide comprehensive insights into OsBgal14's biological role in rice reproductive development .