Orthologs of the Arabidopsis CLAVATA1 Gene in the Cultivated Brassicaceae Plants

In Arabidopsis thaliana, the CLAVATA1 (CLV1) gene is involved in maintaining the balance between the stem cells in the central zone of the stem apical meristem and the determined cells at its periphery. However, CLV1 has not been previously characterized in other Brassicaceae. Using the direct amplification of genomic DNA, we obtained a full-length CLV1 ortholog from canola plants (Brassica napus), and also three CLV1 fragments from rape (B. rapa), canola (B. napus), and false flax (Camelina sativa), which corresponded to the transmembrane domain and a part of the kinase domain of the CLAVATA1 protein. The nucleotide and deduced amino acid sequences of the full-size CLV1 ortholog from B. napus were similar by 81 and 87% to the prototype gene from arabidopsis; in the case of shorter gene fragments, the similarity was as high as 91–93 and 98%, respectively. By their primary structure, the CLV1 genes in the Brassicaceae considerably differ from their putative structural homologs beyond this family.     

The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase.

The CLAVATA2 (CLV2) gene regulates both meristem and organ development in Arabidopsis. We isolated the CLV2 gene and found that it encodes a receptor-like protein (RLP), with a presumed extracellular domain composed of leucine-rich repeats similar to those found in plant and animal receptors, but with a very short predicted cytoplasmic tail. RLPs lacking cytoplasmic signaling domains have not been previously shown to regulate development in plants. Our prior work has demonstrated that the CLV1 receptor-like kinase (RLK) is present as a disulfide-linked multimer in vivo. We report that CLV2 is required for the normal accumulation of CLV1 protein and its assembly into protein complexes, indicating that CLV2 may form a heterodimer with CLV1 to transduce extracellular signals. Sequence analysis suggests that the charged residue in the predicted transmembrane domain of CLV2 may be a common feature of plant RLPs and RLKs. In addition, the chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism, with 50 nucleotide and 15 amino acid differences between Landsberg erecta and Columbia ecotypes within the CLV2 coding sequence.     

Survey of aliphatic glucosinolates in Sicilian wild and cultivated Brassicaceae

In the frame of the activities carried out to exploit Sicilian local cultivars of brassicas, we focused our attention on some of the potential health compounds of various local cruciferous crops. These compounds are of interest to improve the quality of the produce with the aim to develop new cultivars capable of providing functional foods able to prevent disease. In this context, we surveyed for the presence of specific glucosinolates in local cultivars of broccoli, cauliflower, kale, and in some wild species widespread in Sicily, using as control various commercial cultivars. Glucosinolate composition varied extensively among species and crops of the same species, such as cauliflower, broccoli and kale. Cultivar variation for glucosinolate profile was also observed for some crops. For example, Sicilian cultivars of cauliflower possessing colored curds displayed a high content of glucosinolates, glucoraphanin in particular, compared to white curd commercial cultivars. Also some wild species had a high content of other glucosinolates.     

Reproduction during spaceflight by plants in the family Brassicaceae

Researchers report on studies of reproduction in Arabidopsis thaliana in space during during the Chromex-03 on STS-54, Chromex-04 on STS-51, and Chromex-05 on STS-68 missions. The obstacles to seed formation were related to carbon dioxide levels. Other experiments examined in flight pollination and seed production in Brassica rapa during parabolic flight, a 4-1/2 month stay on Mir, and on STS-87. During the Mir experiment, Brassica seeds were harvested from seeds sown in flight. The second generation seeds grew to produce new seeds that contained more starch and less protein and lipid when compared to ground control seeds.     

HOS1, a genetic locus involved in cold-responsive gene expression in arabidopsis.

Low-temperature stress induces the expression of a variety of genes in plants. However, the signal transduction pathway(s) that activates gene expression under cold stress is poorly understood. Mutants defective in cold signaling should facilitate molecular analysis of plant responses to low temperature and eventually lead to the identification and cloning of a cold stress receptor(s) and intracellular signaling components. In this study, we characterize a plant mutant affected in its response to low temperatures. The Arabidopsis hos1-1 mutation identified by luciferase imaging causes superinduction of cold-responsive genes, such as RD29A, COR47, COR15A, KIN1, and ADH. Although these genes are also induced by abscisic acid, high salt, or polyethylene glycol in addition to cold, the hos1-1 mutation only enhances their expression under cold stress. Genetic analysis revealed that hos1-1 is a single recessive mutation in a nuclear gene. Our studies using the firefly luciferase reporter gene under the control of the cold-responsive RD29A promoter have indicated that cold-responsive genes can be induced by temperatures as high as 19 degrees C in hos1-1 plants. In contrast, wild-type plants do not express the luciferase reporter at 10 degrees C or higher. Compared with the wild type, hos1-1 plants are l ess cold hardy. Nonetheless, after 2 days of cold acclimation, hos1-1 plants acquired the same degree of freezing tolerance as did the wild type. The hos1-1 plants flowered earlier than did the wild-type plants and appeared constitutively vernalized. Taken together, our findings show that the HOS1 locus is an important negative regulator of cold signal transduction in plant cells and that it plays critical roles in controlling gene expression under cold stress, freezing tolerance, and flowering time.     

十字花科植物FAE1基因的克隆与功能验证

对十字花科(Brassicaceae)植物非洲芥菜(Brassica tournefortii Gouan)、埃塞俄比亚芥(B.carinataA.Braun)、短喙芥(B.elongata Ehrhart)、芝麻菜[Eruca vesicaria subsp.sativa(Miller) Thellung]、野萝卜(Raphanus raphanistrum Linn.)、Crambe filiformis Jacq.、菥蓂(Thlaspi arvense Linn.)、臭荠[Coronopus didymus (Linn.) Smith]、荠[Capsella bursa-pastoris(Linn.) Medikus]和小花碎米荠(Cardamine parviflora Linn.)的FAE1基因进行了克隆、序列比对及功能验证.结果显示:上述前6种1亚种的FAE1基因长度均为1 521 bp,臭荠的FAE1基因长度为1 517 bp,荠和小花碎米荠的FAE1基因长度为1 518 bp,GenBank登录号为JX898749-JX898758;它们的序列相似性较高,相似度达89％;对位排列矩阵长度1 521 bp,其中包含保守位点1 051个(69.1％)、变异位点470个(30.9％)和简约信息位点232个(15.3％);臭荠、荠和小花碎米荠的FAE1序列在第132位分别缺失3个碱基,臭荠的FAE1基因在第515位缺失1个碱基.虽然荠和小花碎米荠的FAE1基因编码505个氨基酸、臭荠的FAE1基因仅编码186个氨基酸、其他种类的FAE1基因均编码506个氨基酸,但它们的氨基酸序列相似度高达88.9％;各种类的氨基酸序列存在151个变异位点,其中有6个变异位点与种子芥酸含量相关.Western blot及气相色谱分析结果表明:各种类的FAE1基因在酵母中均能表达出预期的蛋白产物;在臭荠和小花碎米荠FAE1基因的转化酵母细胞中无芥酸积累,而在其他种类FAE1基因的转化酵母细胞中均有芥酸积累;此外,除荠外的其他8种1亚种植物的种子芥酸含量与转化酵母细胞中的芥酸含量正相关.     

The receptor kinase CORYNE of Arabidopsis transmits the stem cell-limiting signal CLAVATA3 independently of CLAVATA1

Stem cells in shoot and floral meristems of Arabidopsis thaliana secrete the signaling peptide CLAVATA3 (CLV3) that restricts stem cell proliferation and promotes differentiation. The CLV3 signaling pathway is proposed to comprise the receptor kinase CLV1 and the receptor-like protein CLV2. We show here that the novel receptor kinase CORYNE (CRN) and CLV2 act together, and in parallel with CLV1, to perceive the CLV3 signal. Mutations in CRN cause stem cell proliferation, similar to clv1, clv2, and clv3 mutants. CRN has additional functions during plant development, including floral organ development, that are shared with CLV2. The CRN protein lacks a distinct extracellular domain, and we propose that CRN and CLV2 interact via their transmembrane domains to establish a functional receptor.     

LRP1, a gene expressed in lateral and adventitious root primordia of arabidopsis.

We describe a gene that is expressed in lateral and adventitious root primordia of Arabidopsis. The gene was identified by expression of a transposon-borne promoterless beta-glucuronidase gene in lateral root primordia. The gene, designated LRP1 for lateral root primordium 1, and its corresponding cDNA were cloned and sequenced. The expression pattern of the gene in lateral root primordia was confirmed by in situ hybridization with LRP1 cDNA probes. The LRP1 gene encodes a novel protein. LRP1 expression is activated during the early stages of root primordium development and is turned off prior to the emergence of lateral roots from the parent root. Insertion of the transposon in the LRP1 gene disrupted its expression. To evaluate the homozygous insertion line for a mutant phenotype, several aspects of wild-type lateral root development were analyzed. A mutant phenotype has not yet been identified in the insertion line; however, there is evidence that the gene belongs to a small gene family. LRP1 provides a molecular marker to study the early stages of lateral and adventitious root primordium development.     

The classical arabinogalactan protein gene family of arabidopsis

Arabinogalactan proteins (AGPs) are extracellular proteoglycans implicated in plant growth and development. We searched for classical AGPs in Arabidopsis by identifying expressed sequence tags based on the conserved domain structure of the predicted protein backbone. To confirm that these genes encoded bona fide AGPs, we purified native AGPs and then deglycosylated and deblocked them for N-terminal protein sequencing. In total, we identified 15 genes encoding the protein backbones of classical AGPs, including genes for AG peptides-AGPs with very short backbones (10 to 13 amino acid residues). Seven of the AGPs were verified as AGPs by protein sequencing. A gene encoding a putative cell adhesion molecule with AGP-like domains was also identified. This work provides a firm foundation for beginning functional analysis by using a genetic approach.     

Salicylic acid and NIM1/NPR1-independent gene induction by incompatible Peronospora parasitica in arabidopsis

To identify pathogen-induced genes distinct from those involved in systemic acquired resistance, we used cDNA-amplified fragment length polymorphism to examine RNA levels in Arabidopsis thaliana wild type, nim1-1, and salicylate hydroxylase-expressing plants after inoculation with an incompatible isolate of the downy mildew pathogen Peronospora parasitica. Fifteen genes are described, which define three response profiles on the basis of whether their induction requires salicylic acid (SA) accumulation and NIM1/NPR1 activity, SA alone, or neither. Sequence analysis shows that the genes include a calcium binding protein related to TCH3, a protein containing ankyrin repeats and potential transmembrane domains, three glutathione S-transferase gene family members, and a number of small, putatively secreted proteins. We further characterized this set of genes by assessing their expression patterns in each of the three plant lines after inoculation with a compatible P. parasitica isolate and after treatment with the SA analog 2,6-dichloroisonicotinic acid. Some of the genes within subclasses showed different requirements for SA accumulation and NIM1/NPR1 activity, depending upon which elicitor was used, indicating that those genes were not coordinately regulated and that the regulatory pathways are more complex than simple linear models would indicate.     

Overexpression of seagrass DnaJ gene ZjDjB1 enhances the thermotolerance of transgenic arabidopsis thaliana

Seagrass meadows are one of the most important marine resources that grow along the coast. They provide habitat and a food source for animals. They also protect the coast, fix sediment and purify seawater. In the current period of global climate change, anomalies in coastal water temperatures are increasing. A sudden increase in water temperature owing to a heat wave can have a profound effect on seagrass. Zostera japonica is a type of intertidal seagrasses, which is exposed to the air at low tide. High temperatures in the summer often lead to a decline in seagrass meadows. DnaJ proteins, also known as J proteins, are a family of conserved chaperone proteins. They are designated as J proteins because they contain a highly conserved J domain. They function as chaperones of heat shock proteins in organisms. In this study, the role of DnaJ protein (ZjDjB1) of Z. japonica under heat stress was studied. ZjDjB1 was localized to the cytoplasm and nucleus. The overexpression of ZjDjB1 in Arabidopsis thaliana results in an increase in thermotolerance and a decrease in the accumulation of reactive oxygen species and also a reduction in membrane damage. ZjDjB1 may achieve this goal by maintaining a low activity of proteolytic enzymes.     

Abnormal callose response phenotype and hypersusceptibility to Peronospoara parasitica in defence-compromised arabidopsis nim1-1 and salicylate hydroxylase-expressing plants

To investigate the impact of induced host defenses on the virulence of a compatible Peronospora parasitica strain on Arabidopsis thaliana, we examined growth and development of this pathogen in nim1-1 mutants and transgenic salicylate hydroxylase plants. These plants are unable to respond to or accumulate salicylic acid (SA), respectively, are defective in expression of systemic acquired resistance (SAR), and permit partial growth of some normally avirulent pathogens. We dissected the P. parasitica life cycle into nine stages and compared its progression through these stages in the defense-compromised hosts and in wild-type plants. NahG plants supported the greatest accumulation of pathogen biomass and conidiophore production, followed by nim1-1 and then wild-type plants. Unlike the wild type, NahG and nim1-1 plants showed little induction of the SAR gene PR-1 after colonization with P parasitica, which is similar to our previous observations. We examined the frequency and morphology of callose deposits around parasite haustoria and found significant differences between the three hosts. NahG plants showed a lower fraction of haustoria surrounded by thick callose encasements and a much higher fraction of haustoria with callose limited to thin collars around haustorial necks compared to wild type, whereas nim1-1 plants were intermediate between NahG and wild type. Chemical induction of SAR in plants colonized by P. parasitica converted the extrahaustorial callose phenotype in NahG to resemble closely the wild-type pattern, but had no effect on nim1-1 plants. These results suggest that extrahaustorial callose deposition is influenced by the presence or lack of SA and that this response may be sensitive to the NIM1/NPR1 pathway. Additionally, the enhanced susceptibility displayed by nim1-1 and NahG plants shows that even wild-type susceptible hosts exert defense functions that reduce disease severity and pathogen fitness.     

The arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway

delayed dehiscence1 is an Arabidopsis T-DNA mutant in which anthers release pollen grains too late for pollination to occur. The delayed dehiscence1 defect is caused by a delay in the stomium degeneration program. The gene disrupted in delayed dehiscence1 encodes 12-oxophytodienoate reductase, an enzyme in the jasmonic acid biosynthesis pathway. We rescued the mutant phenotype by exogenous application of jasmonic acid and obtained seed set from previously male-sterile plants. In situ hybridization studies showed that during the early stages of floral development, DELAYED DEHISCENCE1 mRNA accumulated within all floral organs. Later, DELAYED DEHISCENCE1 mRNA accumulated specifically within the pistil, petals, and stamen filaments. DELAYED DEHISCENCE1 mRNA was not detected in the stomium and septum cells of the anther that are involved in pollen release. The T-DNA insertion in delayed dehiscence1 eliminated both DELAYED DEHISCENCE1 mRNA accumulation and 12-oxophytodienoate reductase activity. These experiments suggest that jasmonic acid signaling plays a role in controlling the time of anther dehiscence within the flower.     

Second-generation sequencing for gene discovery in the Brassicaceae

The Brassicaceae contains the most diverse collection of agriculturally important crop species of all plant families. Yet, this is one of the few families that do not form functional symbiotic associations with mycorrhizal fungi in the soil for improved nutrient acquisition. The genes involved in this symbiosis were more recently recruited by legumes for symbiotic association with nitrogen-fixing rhizobia bacteria. This study applied second-generation sequencing (SGS) and analysis tools to discover that two such genes, NSP1 (Nodulation Signalling Pathway 1) and NSP2, remain conserved in diverse members of the Brassicaceae despite the absence of these symbioses. We demonstrate the utility of SGS data for the discovery of putative gene homologs and their analysis in complex polyploid crop genomes with little prior sequence information. Furthermore, we show how this data can be applied to enhance downstream reverse genetics analyses. We hypothesize that Brassica NSP genes may function in the root in other plant-microbe interaction pathways that were recruited for mycorrhizal and rhizobial symbioses during evolution.     

Water-soluble chlorophyll protein in Brassicaceae plants is a stress-induced chlorophyll-binding protein

Two kinds of water-soluble chlorophyll (Chl) proteins (WSCPs) have been found, e.g., a WSCP from Chenopodium, Atriplex, Polygonum, and Amaranthus species (class I) and that from Brassica, Raphanus, and Lepidium species (class II). Classes I and II WSCPs differ mainly in their photoconvertiblity. Class I WSCPs show a light-induced absorption change, whereas Class II WSCPs do not. The molecular and functional properties of Class I WSCP are largely uncertain. On the other hand, recent studies on the adaptation of plants to osmotic stress revealed the participation of drought-stress induced proteins with molecular masses of 20-22 kDa possessing a sequence similarity with class II WSCPs. This mini review focuses on the molecular signature of class II WSCPs. The physiological function of class II WSCPs has not been clarified either, but, their water-solubility, low Chl content, and stress-inducibility suggested little contribution to photosynthesis. Several molecular properties predicting its physiological role are as follows. The WSCP tetramer, may have only one or no Chl molecules in each subunit. All WSCPs possess a motif for Künitz-type proteinase inhibitor family in their sequence. WSCP is induced by drought- and heat-stresses suggesting its protective role during stress conditions. Monomeric recombinant apo-WSCP is able to remove Chls from the thylakoid membrane in aqueous solution and form into a tetramer. Brassica-WSCP contains a signal sequence targeted to endoplasmic reticulum. The highly conserved, C-terminal region is missing in the mature WSCP. Possible functions of class II WSCPs in plant tissues are discussed.