Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (CONSTANS, CONSTANS-LIKE and TIMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha-albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.

Accumulation of Phenolic Compounds and Glucosinolates in Sprouts of Pale Green and Purple Kohlrabi (Brassica oleracea var. gongylodes) under Light and Dark Conditions

Kohlrabi is considered an important dietary vegetable worldwide. In this study, we investigated the growth and accumulation of phenolic compounds (PCs) and glucosinolates in sprouts of pale green and purple kohlrabi (Brassica oleracea var. gongylodes) in response to light and dark conditions. Pale green kohlrabi presented high fresh weight and root length irrespective of light treatment, whereas under dark conditions, it presented higher fresh weight and shoot length than purple kohlrabi. In contrast, the root length of both kohlrabies increased markedly under light conditions compared to that under dark conditions. Thirteen PCs and eight glucosinolates were detected and quantified in 10-day-old pale green and purple kohlrabies. In both kohlrabies, the individual and total phenolic levels were much higher under the light treatment than under the dark treatment. Under light and dark conditions, the total phenolic content was 6362.13 and 5475.04 µg/g dry weight in the pale green kohlrabi, respectively, whereas in the purple kohlrabi, it was 10,115.76 and 9361.74 µg/g dry weight, respectively. Dark conditions favored higher accumulation of glucosinolates than light conditions. Progoitrin, neoglucobrassicin, glucoerucin, and 4-methoxyglucobrassicin were the predominant glucosinolates in both kohlrabies and were present in much higher amounts in the pale green kohlrabi. In pale green kohlrabi under dark conditions, the total glucosinolates content was 4.75 and 2.62 times higher than that of the purple kohlrabi under light and dark conditions, respectively. Among individual glucosinolates, in the pale green kohlrabi under the dark condition, progoitrin was found to have the highest content, which was 90.28 and 54.51 times higher than that in the purple kohlrabi under light and dark conditions, respectively. These results show that the phenolic and glucosinolates levels varied widely, and these variations between the two types of kohlrabi under both light and dark conditions were significant. Our findings suggest that light and dark conditions enhance the accumulation of PCs and glucosinolates, respectively, during the development of kohlrabi seedlings.

Mapping of a Pale Green Mutant Gene and Its Functional Verification by Allelic Mutations in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Leaves are the main organ for photosynthesis, and variations in leaf color affect photosynthesis and plant biomass formation. We created two similar whole-plant pale green mutants (pem1 and pem2) from the double haploid (DH) Chinese cabbage line “FT” through ethyl methanesulfonate (EMS) mutagenesis of seeds. Photosynthetic pigment contents and net photosynthetic rates were significantly lower in the mutants than in the wild-type “FT,” and the chloroplast thylakoid endomembrane system was poor. Genetic analysis showed that the mutated phenotypes of pem1 and pem2 were caused by a single nuclear gene. Allelism tests showed that pem1 and pem2 were alleles. We mapped Brpem1 to a 64.25 kb region on chromosome A10, using BSR-Seq and map-based cloning of 979 F2 recessive individuals. Whole-genome re-sequencing revealed a single nucleotide polymorphism (SNP) transition from guanine to adenosine on BraA10g021490.3C in pem1, causing an amino acid shift from glycine to glutamic acid (G to E); in addition, BraA10g021490.3C in pem2 was found to have a single nucleotide substitution from guanine to adenosine, causing an amino acid change from E to lysine (K). BraA10g021490.3C is a homolog of the Arabidopsisdivinyl chlorophyllide a 8-vinyl-reductase (DVR) gene that encodes 3,8-divinyl protochlorophyllide a 8-vinyl reductase, which is a key enzyme in chlorophyll biosynthesis. Enzyme activity assay and chlorophyll composition analysis demonstrated that impaired DVR had partial loss of function. These results provide a basis to understand chlorophyll metabolism and explore the mechanism of a pale green phenotype in Chinese cabbage.

BrRNE cleaves RNA in chloroplasts, regulating retrograde signals in Brassica rapa L. ssp. pekinensis

Key message Brassica rapa RNE participates in the processing of polycistronic precursor transcripts into mature monocistronic mRNAs in plastids, thereby sending strong retrograde signals. Abstract Leaf color is one of the most important agronomic traits for Chinese cabbage. Not only is it closely linked to photosynthesis, thereby affecting plant growth, but it also influences consumer preference in the marketplace. A pale-green mutant rne was produced by EMS mutagenesis of Chinese cabbage inbred line A03. Chlorophyll content, photosynthetic rate, actual quantum efficiency (φPSII), and maximum quantum efficiency (Fv/Fm) of photosystem II (PSII) were all reduced in rne plants. Genetic analysis indicated that the pale-green trait was controlled by a pair of recessive alleles. Using mixed pool sequencing of F2 individuals derived from an rne × wild-type cross, we identified the essential gene Brassica rapa RNase E (BrRNE), which is responsible for chloroplast development. BrRNE cleaves polycistronic RNA in Chinese cabbage A03 plastids, but rne plants are defective in RNA processing and show reduced translation levels of the seven plastid genes, BrpsaB, BrpsaA, BrpsbA, BrpsbD, BrpsbB, BrpetA, and Brycf4. Abnormal RNA processing in the plastids sends retrograde signals that markedly regulate the expression of nuclear genes, upregulating genes that participate in ribosome and DNA replication pathways and repressing photosynthesis-associated nuclear genes (PhANGs). Our study reveals a new regulatory mechanism by which plastid RNA cleavage influences plastid development and leaf color, sending retrograde signals that affect the expression of nuclear genes in Brassica.

Development of pharmacognostic parameters for the leaf of Bridelia scandens (Roxb.) Willd

Background: Pharmacognostic study of medicinal plants is an important parameter for standardization and authentication of plants, with the help of which adulteration and substitution can be prevented. The present study deals with pharmacognostic profile of leaf of Bridelia scandens (Roxb).Willd. an important traditional plant, belonging to family Euphorbiaceae used to treat various ailments. Methods: The present study includes macroscopic and microscopic studies, quantitative microscopy, and physiochemical characters such as ash value, extractive values, fluorescence analysis, and total phenol and flavonoid content. Results: Macroscopically, the leaves are B. scandens are elliptic –oblong or obavate, dark green above, pale green below lateral veins. Microscopically, leaf consists of thick semicircular midrib and the lamina, cortical zone ending with thick continuous cylinder of sclerenchyma cells. Sclerenchyma cylinder completely enclosed the vascular cylinder of the midrib, consists of only continuous thick layer of phloem. Xylem cylinder consists of numerous short or long radial chains of vessels. The lateral vein is flat on the adaxial side and prominently projecting hemispherical body on the adaxial side. Powder microscopy of leaf revealed the presence of spiral xylem vessels, rosette and prismatic calcium oxalate crystals and trichomes. A Physiochemical characteristic was also determined. Conclusion: Existing literature revealed that so far, no Pharmacognostic study has been reported on the leaf of B. scandens. Findings from this investigation can be used for its identification and determination of quality and purity of medicinally important plant. Thus exploring the usefulness of pharmacognostic evaluation to validate and authenticate drug

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs Involved in Chloroplast Development

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

A new karst-dwelling, colorful pitviper (Viperidae: Trimeresurus) from northern Peninsular Thailand

We describe a colorful and distinctively patterned, karst-dwelling pitviper, Trimeresurus kuiburi sp. nov., from the isolated, coastal massif of Khao Sam Roi Yot in Kui Buri District, Prachuap Khiri Khan Province, in northern Peninsular Thailand. The new species, member of the ‘Cryptelytrops group’ sensu Malhotra & Thorpe (2004) and morphologically and genetically allied to Trimeresurus kanburiensis and T. venustus, differs from all pitviper taxa by a combination of red/purple bands on a green dorsum; a white concave suborbital stripe in males (straight and less visible in females); white, spaced vertebral dots in males (absent in females); pale green belly lacking dark dots or stripe on the lateral sides of the ventrals; partially fused first supralabial and nasal scale; 19 dorsal scale rows at midbody; 164–171 ventrals; 63–65 subcaudals in males, 51–53 in females; maximal known SVL of 451 mm; and long, papillose hemipenes.

Begonia rigidifolia ssp. sonhungii (sect. Petermannia, Begoniaceae), a new subspecies from limestone hills in Central Vietnam

Begonia rigidifolia Aver. ssp. sonhungii C.W.Lin (sect. Petermannia, Begoniaceae) from Central Vietnam is hereby described and illustrated as new. It resembles B. rigidifolia ssp. rigidifolia, but differs in its very sparsely puberulous (vs. glabrous), darker toned lamina that is embellished with a pale green broad band (vs. emerald green lamina with irregular radiantly arranged dark spots), stamens widely obovate (vs. obovate) and capsule with wider abaxial wing 6–10 mm (vs. 2–3 mm).

An Argon-Ion-Induced Pale Green Mutant of Arabidopsis Exhibiting Rapid Disassembly of Mesophyll Chloroplast Grana

Argon-ion beam is an effective mutagen capable of inducing a variety of mutation types. In this study, an argon ion-induced pale green mutant of Arabidopsis thaliana was isolated and characterized. The mutant, designated Ar50-33-pg1, exhibited moderate defects of growth and greening and exhibited rapid chlorosis in photosynthetic tissues. Fluorescence microscopy confirmed that mesophyll chloroplasts underwent substantial shrinkage during the chlorotic process. Genetic and whole-genome resequencing analyses revealed that Ar50-33-pg1 contained a large 940 kb deletion in chromosome V that encompassed more than 100 annotated genes, including 41 protein-coding genes such as TYRAAt1/TyrA1, EGY1, and MBD12. One of the deleted genes, EGY1, for a thylakoid membrane-localized metalloprotease, was the major contributory gene responsible for the pale mutant phenotype. Both an egy1 mutant and F1 progeny of an Ar50-33-pg1 × egy1 cross-exhibited chlorotic phenotypes similar to those of Ar50-33-pg1. Furthermore, ultrastructural analysis of mesophyll cells revealed that Ar50-33-pg1 and egy1 initially developed wild type-like chloroplasts, but these were rapidly disassembled, resulting in thylakoid disorganization and fragmentation, as well as plastoglobule accumulation, as terminal phenotypes. Together, these data support the utility of heavy-ion mutagenesis for plant genetic analysis and highlight the importance of EGY1 in the structural maintenance of grana in mesophyll chloroplasts.