A pair of DUF538 domain-containing proteins modulates plant growth and trichome development through the transcriptional regulation of GLABRA1 in Arabidopsis thaliana.

SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) is an emerging plant growth regulator in trichome development, endoplasmic reticulum stress response, and phosphoinositide signaling, and belongs to the land plant-specific DUF538 domain-containing protein family. Despite its multifaceted roles, the functions of this protein family are poorly understood in plant growth and development. Here, we report that SVB-like (SVBL), the closest homolog of SVB, modulates plant growth and trichome development with SVB in Arabidopsis thaliana. Although none of the single mutants showed an obvious growth defect, the double mutants of svb svbl exhibited dwarfed plant growth. In trichome development, the defects in svb mutant were greatly enhanced by the additional mutation in SVBL, despite the single knockout of SVBL showing the mild defects. The double mutation reduced the transcript level of one of the central hub genes for trichome development, GLABRA1 (GL1), which in turn affects the other downstream genes, GLABRA2 (GL2), TRANSPARENT TESTA GLABRA2 (TTG2), TRIPTYCHON (TRY), CAPRICE (CPC), and ENHANCER OF TRY AND CPC1 (ETC1). In situ translational reporter assays showed that SVB and SVBL share highly similar localization patterns both at tissue and subcellular levels. The present study suggests that SVB and SVBL play a pivotal role in plant growth and trichome development by affecting a specific subset of known trichome developmental regulators, highlighting the importance of the DUF538 protein family in higher plants.

What's unique? The unfolded protein response in plants.

The investigation of a phenomenon called the unfolded protein response (UPR) started approximately three decades ago, and we now know that the UPR is involved in a number of cellular events among metazoans, higher plants, and algae. The relevance of the UPR in human diseases featuring protein folding defects, such as Alzheimer's and Huntington's diseases, has drawn much attention to the response in medical research to date. While metazoans and plants share similar molecular mechanisms of the UPR, recent studies shed light on the uniqueness of the plant UPR, with plant-specific protein families appearing to play pivotal roles. Given the considerable emphasis on the original discoveries of key factors in metazoans, this review highlights the uniqueness of the plant UPR based on current knowledge.

Pectin methylesterase activity of plant DUF538 protein superfamily.

DUF538 (domain of unknown function 538) proteins are known as a group of putative hypothetical proteins in a wide range of plant species. They have been identified from some plants challenged with various environmental stresses. However, a little is known about their functional properties. They have been newly predicted to have binding capacity and esterase-type hydrolytic activity towards bacterial lipopolysaccharides and chlorophyll molecules as carboxylic compounds in plants. In the present study, the binding ability and the methylesterase activity of DUF538 proteins towards pectin molecules were also predicted. Their similarities to pectin methylesterases and their binding ability to pectin molecule were predicted using bioinformatic tools as well as the experimental method. A probable cooperation was speculated between DUF538 and pectin methylesterase protein families in cell wall associated defense responses in plants.

Maize glossy6 is involved in cuticular wax deposition and drought tolerance.

Cuticular waxes, long-chain hydrocarbon compounds, form the outermost layer of plant surfaces in most terrestrial plants. The presence of cuticular waxes protects plants from water loss and other environmental stresses. Cloning and characterization of genes involved in the regulation, biosynthesis, and extracellular transport of cuticular waxes onto the surface of epidermal cells have revealed the molecular basis of cuticular wax accumulation. However, intracellular trafficking of synthesized waxes to the plasma membrane for cellular secretion is poorly understood. Here, we characterized a maize glossy (gl6) mutant that exhibited decreased epicuticular wax load, increased cuticle permeability, and reduced seedling drought tolerance relative to wild-type. We combined an RNA-sequencing-based mapping approach (BSR-Seq) and chromosome walking to identify the gl6 candidate gene, which was confirmed via the analysis of multiple independent mutant alleles. The gl6 gene represents a novel maize glossy gene containing a conserved, but uncharacterized, DUF538 domain. This study suggests that the GL6 protein may be involved in the intracellular trafficking of cuticular waxes, opening the door to elucidating the poorly understood process by which cuticular wax is transported from its site of biosynthesis to the plasma membrane.

DUF538 protein superfamily is predicted to be chlorophyll hydrolyzing enzymes in plants.

The possible hydrolytic activity towards chlorophyll molecules was predicted for DUF538 protein superfamily in plants. It was examined by using computational as well as experimental tools including in vitro chlorophyll degradation, antioxidant compounds production and in vivo real-time gene expression tests. Comparison of the computational data with the experimental results indicated that DUF538 proteins might be chlorophyll hydrolyzing enzyme (most probably carboxyesterase) which degrade chlorophyll molecules (66 % per 12 hrs) to produce new compounds (1.8 fold per 12 hrs) with antioxidant properties. The relevance of DUF538 gene expression level with the chlorophyll contents (2.8 fold increase per chlorophyll content of 50 %) of the drought-stressed leaves showed that chlorophyll degradation by DUF538 is most probably induced in response to stress stimuli. Despite membranous chlorophyll catabolic pathways, DUF538-dependent reactions is predicted to be occurred in the cytosol of the under stressed plants. We addressed as to whether chlorophyll breakdown to antioxidant compounds by DUF538 is a defense mechanism of plants against stress stimuli, in vivo? This question is going to be investigated in our next research project.

Effect of near-infrared irradiation on photoconversion of the water-soluble chlorophyll-binding protein of Chenopodium album.

We investigated the effects of near-infrared irradiation on the photoconversion of Chenopodium album water-soluble chlorophyll-binding protein (CaWSCP) in the presence of sodium hydrosulfite and found a further photoconversion from CP742 to CP763, a novel form of CaWSCP. Interestingly, one-third of the absorption peak at 668 nm was recovered in CP763, but re-irradiation under oxidative conditions eliminated the photo convertibility of CaWSCP.

Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium.

Photoconvertible water-soluble chlorophyll-binding proteins, called Class I WSCPs, have been detected in Chenopodiaceae, Amaranthaceae and Polygonaceae plant species. To date, Chenopodium album WSCP (CaWSCP) is the only cloned gene encoding a Class I WSCP. In this study, we identified two cDNAs encoding Chenopodium ficifolium Class I WSCPs, CfWSCP1, and CfWSCP2. Sequence analyses revealed that the open reading frames of CfWSCP1 and CfWSCP2 were 585 and 588 bp, respectively. Furthermore, both CfWSCPs contain cystein2 and cystein30, which are essential for the chlorophyll-binding ability of CaWSCP. Recombinant CfWSCP1 and CfWSCP2, expressed in Escherichia coli as hexa-histidine fusion proteins (CfWSCP1-His and CfWSCP2-His), formed inclusion bodies; however, we were able to solubilize these using a buffer containing 8 M urea and then refold them by dialysis. The refolded CfWSCP1-His and CfWSCP2-His could bind chlorophylls and exhibited photoconvertibility, confirming that the cloned CfWSCPs are further examples of Class I WSCPs.

Cysteine-2 and Cys30 are essential for chlorophyll-binding activity of the water-soluble chlorophyll-binding protein (WSCP) of Chenopodium album.

Chenopodium album has a non-photosynthetic chlorophyll protein known as the water-soluble chlorophyll (Chl)-binding protein (WSCP). The C. album WSCP (CaWSCP) is able to photoconvert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton. Reducing reagents such as ß-mercaptoethanol or dithiothreitol inhibit photoconversion, indicating that S-S bridge(s) in CaWSCP are quite important for it. Recently, we found that the mature region of CaWSCP contains five cysteine residues; Cys2, Cys30, Cys48, Cys63, and Cys144. To identify which cysteine residues are involved in the photoconversion, we generated five mutants (C2S, C30S, C48S, C63S, and C144S) by site-directed mutagenesis. Interestingly, C48S, C63S, and C144S mutants showed the same Chl-binding activity and photoconvertibility as those of the recombinant wild-type CaWSCP-His, while the C2S and C30S mutants completely lost Chl-binding activity. Our findings indicated that the S-S bridge between Cys2 and Cys30 in each CaWSCP subunit is essential for Chl-binding activity.

Antagonistic Regulation of ABA Responses by Duplicated Tandemly Repeated DUF538 Protein Genes in Arabidopsis.

The plant hormone ABA (abscisic acid) regulates plant responses to abiotic stresses by regulating the expression of ABA response genes. However, the functions of a large portion of ABA response genes have remained unclear. We report in this study the identification of ASDs (ABA-inducible signal peptide-containing DUF538 proteins), a subgroup of DUF538 proteins with a signal peptide, as the regulators of plant responses to ABA in Arabidopsis. ASDs are encoded by four closely related DUF538 genes, with ASD1/ASD2 and ASD3/ASD4 being two pairs of duplicated tandemly repeated genes. The quantitative RT-PCR (qRT-PCR) results showed that the expression levels of ASDs increased significantly in response to ABA as well as NaCl and mannitol treatments, with the exception that the expression level of ASD2 remained largely unchanged in response to NaCl treatment. The results of Arabidopsis protoplast transient transfection assays showed that ASDs were localized on the plasma membrane and in the cytosol and nucleus. When recruited to the promoter of the reporter gene via a fused GD domain, ASDs were able to slightly repress the expression of the co-transfected reporter gene. Seed germination and cotyledon greening assays showed that ABA sensitivity was increased in the transgenic plants that were over-expressing ASD1 or ASD3 but decreased in the transgenic plants that were over-expressing ASD2 or ASD4. On the other hand, ABA sensitivity was increased in the CRISPR/Cas9 gene-edited asd2 single mutants but decreased in the asd3 single mutants. A transcriptome analysis showed that differentially expressed genes in the 35S:ASD2 transgenic plant seedlings were enriched in several different processes, including in plant growth and development, the secondary metabolism, and plant hormone signaling. In summary, our results show that ASDs are ABA response genes and that ASDs are involved in the regulation of plant responses to ABA in Arabidopsis; however, ASD1/ASD3 and ASD2/ASD4 have opposite functions.

Are tyrosine residues involved in the photoconversion of the water-soluble chlorophyll-binding protein of Chenopodium album?

Non-photosynthetic and hydrophilic chlorophyll (Chl) proteins, called water-soluble Chl-binding proteins (WSCPs), are distributed in various species of Chenopodiaceae, Amaranthaceae, Polygonaceae and Brassicaceae. Based on their photoconvertibility, WSCPs are categorised into two classes: Class I (photoconvertible) and Class II (non-photoconvertible). Chenopodium album WSCP (CaWSCP; Class I) is able to convert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton under light in the presence of molecular oxygen. Potassium iodide (KI) is a strong inhibitor of the photoconversion. Because KI attacks tyrosine residues in proteins, tyrosine residues in CaWSCP are considered to be important amino acid residues for the photoconversion. Recently, we identified the gene encoding CaWSCP and found that the mature region of CaWSCP contained four tyrosine residues: Tyr13, Tyr14, Tyr87 and Tyr134. To gain insight into the effect of the tyrosine residues on the photoconversion, we constructed 15 mutant proteins (Y13A, Y14A, Y87A, Y134A, Y13-14A, Y13-87A, Y13-134A, Y14-87A, Y14-134A, Y87-134A, Y13-14-87A, Y13-14-134A, Y13-87-134A, Y14-87-134A and Y13-14-87-134A) using site-directed mutagenesis. Amazingly, all the mutant proteins retained not only chlorophyll-binding activity, but also photoconvertibility. Furthermore, we found that KI strongly inhibited the photoconversion of Y13-14-87-134A. These findings indicated that the four tyrosine residues are not essential for the photoconversion.

The photoconvertible water-soluble chlorophyll-binding protein of Chenopodium album is a member of DUF538, a superfamily that distributes in Embryophyta.

Various plants possess hydrophilic chlorophyll (Chl) proteins known as water-soluble Chl-binding proteins (WSCPs). WSCPs exist in two forms: Class I and Class II, of which Class I alone exhibits unique photoconvertibility. Although numerous genes encoding Class II WSCPs have been identified and the molecular properties of their recombinant proteins have been well characterized, no Class I WSCP gene has been identified to date. In this study, we cloned the cDNA and a gene encoding the Class I WSCP of Chenopodium album (CaWSCP). Sequence analyses revealed that CaWSCP comprises a single exon corresponding to 585bp of an open reading frame encoding 195 amino acid residues. The CaWSCP protein sequence possesses a signature of DUF538, a protein superfamily of unknown function found almost exclusively in Embryophyta. The recombinant CaWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (CaWSCP-His) that removes Chls from the thylakoid. Under visible light illumination, the reconstituted CaWSCP-His was successfully photoconverted into a different pigment with an absorption spectrum identical to that of native CaWSCP. Interestingly, while CaWSCP-His could bind both Chl a and Chl b, photoconversion occurred only in CaWSCP-His reconstituted with Chl a.