Ectopic expression of soybean GmKNT1 in Arabidopsis results in altered leaf morphology and flower identity

Plant morphology is specified by leaves and flowers, and the shoot apical meristem (SAM) defines the architecture of plant leaves and flowers. Here, we reported the characterization of a soybean KNOX gene GmKNT1, which was highly homologous to Arabidopsis STM. The GmKNT1 was strongly expressed in roots, flowers and developing seeds. Its expression could be induced by IAA, ABA and JA, but inhibited by GA or cytokinin. Staining of the transgenic plants overexpressing GmKNT1-GUS fusion protein revealed that the GmKNT1 was mainly expressed at lobe region, SAM of young leaves, sepal and carpel, not in seed and mature leaves. Scanning electron micros- copy (SEM) disclosed multiple changes in morphology of the epidermal cells and stigma. The transgenic Arabidopsis plants overexpress- ing the GmKNT1 showed small and lobed leaves, shortened internodes and small clustered inflorescence. The lobed leaves might result from the function of the meristems located at the boundary of the leaf. Compared with wild type plants, transgenic plants had higher ex- pression of the SAM-related genes including the CUP, WUS, CUC1, KNAT2 and KNAT6. These results indicated that the GmKNT1 could affect multiple aspects of plant growth and development by regulation of downstream genes expression.     

Eucalyptus has a functional equivalent of the Arabidopsis floral meristem identity gene LEAFY

Two genes cloned from Eucalyptus globulus, Eucalyptus LeaFy (ELF1 and ELF2), have sequence homology to the floral meristem identity genes LEAFY from Arabidopsis and FLORICAULA from Antirrhinum. ELF1 is expressed in the developing eucalypt floral organs in a pattern similar to LEAFY while ELF2 appears to be a pseudo gene. ELF1 is expressed strongly in the early floral primordium and then successively in the primordia of sepals, petals, stamens and carpels. It is also expressed in the leaf primordia and young leaves and adult and juvenile trees.The ELF1 promoter coupled to a GUS reporter gene directs expression in transgenic Arabidopsis in a temporal and tissue-specific pattern similar to an equivalent Arabidopsis LEAFY promoter construct. Strong expression is seen in young flower buds and then later in sepals and petals. No expression was seen in rosette leaves or roots of flowering plants or in any non-flowering plants grown under long days. Furthermore, ectopic expression of the ELF1 gene in transgenic Arabidopsis causes the premature conversion of shoots into flowers, as does an equivalent 35S-LFY construct. These data suggest that ELF1 plays a similar role to LFY in flower development and that the basic mechanisms involved in flower initiation and development in Eucalyptus are similar to those in Arabidopsis.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

Characterization of a Casuarina glauca nodule-specific subtilisin-like protease gene, a homolog of Alnus glutinosa ag12

In search of plant genes expressed during early interactions between Casuarina glauca and Frankia, we have isolated and characterized a C. glauca gene that has strong homology to subtilisin-like protease gene families of several plants including the actinorhizal nodulin gene ag12 of another actinorhizal plant, Alnus glutinosa. Based on the expression pattern of cg12 in the course of nodule development, it represents an early actinorhizal nodulin gene. Our results suggest that subtilisin-like proteases may be a common element in the process of infection of plant cells by Frankia in both Betulaceae (Alnus glutinosa) and Casuarinaceae (Casuarina glauca) symbioses.     

Localization and control of expression of Nt-Syr1, a tobacco SNARE protein

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion and secretion. Previously, we isolated the syntaxin-related protein Nt-Syr1 from Nicotiana in a screen for ABA-related signalling elements, and demonstrated its role in determining the ABA sensitivity of stomatal guard cells. Because the location and expression of SNAREs are often important clues to their functioning, we have examined the distribution and stimulus-dependent expression of Nt-Syr1 between tissues, as well as its location within the cell, using antisera raised against purified recombinant peptides corresponding to overlapping cytosolic domains of Nt-Syr1. The Nt-Syr1 epitope was strongly represented in roots and to lesser extents in stems, leaves and flowers of well-watered plants. Biochemical analysis and examination of immunogold labelling under the electron microscope indicated Nt-Syr1 to be located primarily at the plasma membrane. Expression of the protein in leaves and to a lesser extent in flowers and stems was transiently enhanced by ABA, but not by auxin, kinetin or gibberellic acid. Expression in leaves was promoted by salt stress and wounding, but not by cold. By contrast, Nt-Syr1 levels in the root were unaffected by ABA. In the leaves, enhanced expression of Nt-Syr1 by salt stress was not observed in aba1 mutant Nicotiana, which is deficient in ABA synthesis, and in plants carrying the Arabidopsis abi1 transgene that suppresses a number of ABA-evoked responses in these plants. However, an enhanced expression in response to wounding was observed, even in the mutant backgrounds. We conclude that Nt-Syr1 expression at the plasma membrane is important for its function and is subject to control by parallel, stress-related signalling pathways, both dependent on and independent of ABA. Nt-Syr1 may be associated with additional functions, especially in the roots, that are unrelated to ABA or stress responses in the plant.     

Influence of protein kinase KIN10 gene expression on root phenotype of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> root system under condition of energy stress

The phenotypic changes in the root system of Arabidopsis thaliana seedlings in transgenic lines with overexpression and suppressed gene expression of serine-threonine protein kinase KIN10, under conditions of energy shortage and under normal conditions, were shown. The normal growth and development of KIN10 overexpressing plants under energy deficiency conditions were detected. The significant inhibition of the development of these plant lines was observed under normal conditions. The levels of KIN10 gene expression under normal conditions in different organs of Arabidopsis thaliana, particularly in the roots, stems, leaves and flowers were analyzed. The highest-level expression of the gene was found in the leaves.     

Characterization of the extracellular gamma-glutamyl transpeptidases, GGT1 and GGT2, in Arabidopsis

gamma-Glutamyl transpeptidase (GGT) is the only enzyme known that can cleave the gamma-peptide bond between glutamate and cysteine in glutathione, and is therefore a key step in glutathione degradation. There are three functional GGT genes in Arabidopsis, two of which are considered here. GGT1 and GGT2 are apoplastic, associated with the plasma membrane and/or cell wall. RNA blots and analysis of enzyme activity in knockout mutants suggest that GGT1 is expressed most strongly in leaves but is found throughout the plant. A GGT1::GUS fusion construct showed expression only in vascular tissue, specifically the phloem of the mid-rib and minor veins of leaves, roots and flowers. This localization was confirmed in leaves by laser microdissection. GGT2 expression is limited to embryo, endosperm, outer integument, and a small portion of the funiculus in developing siliques. The ggt2 mutants had no detectable phenotype, while the ggt1 knockouts were smaller and flowered sooner than wild-type. In ggt1 plants, the cotyledons and older leaves yellowed early, and GSSG, the oxidized form of glutathione, accumulated in the apoplastic space. These observations suggest that GGT1 is important in preventing oxidative stress by metabolizing extracellular GSSG, while GGT2 might be important in transporting glutathione into developing seeds.     

A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants.

The surfaces of land plants are covered with a cuticle that is essential for retention of water. Epidermal surfaces of Arabidopsis thaliana embryos and juvenile plants that were homozygous for abnormal leaf shape1 (ale1) mutations were defective, resulting in excessive water loss and organ fusion in young plants. In ale1 embryos, the cuticle was rudimentary and remnants of the endosperm remained attached to developing embryos. Juvenile plants had a similar abnormal cuticle. The ALE1 gene was isolated using a transposon-tagged allele ale1-1. The predicted ALE1 amino acid sequence was homologous to those of subtilisin-like serine proteases. The ALE1 gene was found to be expressed within certain endosperm cells adjacent to the embryo and within the young embryo. Expression was not detected after germination. Our results suggest that the putative protease ALE1 affects the formation of cuticle on embryos and juvenile plants and that an appropriate cuticle is required for separation of the endosperm from the embryo and for prevention of organ fusion.     

Tight coupling of root-associated nitrogen fixation and plant photosynthesis in the salt marsh grass Spartina alterniflora and carbon dioxide enhancement of nitrogenase activity

The coupling of root-associated nitrogen fixation and plant photosynthesis was examined in the salt marsh grass Spartina alterniflora. In both field experiments and hydroponic assay chambers, nitrogen fixation associated with the roots was rapidly enhanced by stimulating plant photosynthesis. A kinetic analysis of acetylene reduction activity (ARA) showed that a five-to sixfold stimulation occurred within 10 to 60 min after the plant leaves were exposed to light or increased CO2 concentrations (with the light held constant). In field experiments, CO2 enrichment increased plant-associated ARA by 27%. Further evidence of the dependence of ARA on plant photosynthate was obtained when activity in excised roots was shown to decrease after young greenhouse plants were placed in the dark. Seasonal variation in the ARA of excised plant roots from field cores appears to be related to the annual cycle of net photosynthesis in S. alterniflora.     

Tight coupling of root-associated nitrogen fixation and plant photosynthesis in the salt marsh grass Spartina alterniflora and carbon dioxide enhancement of nitrogenase activity.

The coupling of root-associated nitrogen fixation and plant photosynthesis was examined in the salt marsh grass Spartina alterniflora. In both field experiments and hydroponic assay chambers, nitrogen fixation associated with the roots was rapidly enhanced by stimulating plant photosynthesis. A kinetic analysis of acetylene reduction activity (ARA) showed that a five-to sixfold stimulation occurred within 10 to 60 min after the plant leaves were exposed to light or increased CO2 concentrations (with the light held constant). In field experiments, CO2 enrichment increased plant-associated ARA by 27%. Further evidence of the dependence of ARA on plant photosynthate was obtained when activity in excised roots was shown to decrease after young greenhouse plants were placed in the dark. Seasonal variation in the ARA of excised plant roots from field cores appears to be related to the annual cycle of net photosynthesis in S. alterniflora.