Production of soybean isoflavone genistein in non-legume plants via genetically modified secondary metabolism pathway

Genetic modification of secondary metabolic pathways to produce desirable natural products is an attractive approach in plant biotechnology. In our study, we attempted to produce a typical soybean isoflavone genistein, a well-known health-promoting metabolite, in non-legume plants via genetic engineering. Both overexpression and antisense suppression strategies were used to manipulate the expression of several genes encoding key enzymes in the flavonoids/isoflavonoids pathway in transgenic tobacco, lettuce, and petunia. Introducing soybean isoflavone synthase (IFS) into these plants, which naturally do not produce isoflavonoids due to a lack of this leguminous enzyme, resulted in genistein biosynthesis in tobacco petals, petunia leaves and petals, and lettuce leaves. In tobacco, when flavanone 3-hydroxylase (F3H) expression was suppressed by its antisense gene while soybean IFS was overexpressed at the same time, genistein yield increased prominently. In addition, overexpression of phenylalanine ammonia-lyase (PAL) also led to an enhanced genistein production in tobacco petals and lettuce leaves in the presence of IFS than in the plants that overexpressed only IFS.     

PhEXPA1, a Petunia hybrida expansin,is involved in cell wall metabolism and in plant architecture specification

Expansins are wall-loosening proteins that induce wall stress relaxation and irreversible wall extension in a pH-dependent manner. Despite a substantial body of work has been performed on the characterization of many expansins genes in different plant species, the knowledge about their precise biological roles during plant development remains scarce. To yield insights into the expansion process in Petunia hybrida, PhEXPA1, an expansin gene preferentially expressed in petal limb, has been characterized. The constitutive overexpression of PhEXPA1 significantly increased expansin activity, cells size and organ dimensions. Moreover, 35S::PhEXPA1 transgenic plants exhibited an altered cell wall polymer composition and a precocious timing of axillary meristem development compared with wild-type plants. These findings supported a previous hypothesis that expansins are not merely structural proteins involved in plant cell wall metabolism but they also take part in many plant development processes. Here, to support this expansins dual role, we discuss about differential cell wall-related genes expressed in PhEXPA1 expression mutants and gradients of altered petunia branching pattern.     

Ectopic Expression of the Pttknl Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm.

A novel knottedl-like homeobox （knox） gene, Pttknl （Populus tremulaxtremuloides knottedl）, isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leafdisc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on the abaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petal color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed.     

Effect of tropospheric ozone on morphological characteristics of Nicotiana tabacum L., Phaseolus vulgaris L. and Petunia×Hybrida L. in ambient air conditions

The cumulative ozone effect on morphological parameters (visible leaf injury, plant height and leaf growth, number of bean pods, petunia flowers and stalks) was examined in this study. Well-known ozonesensitive (Bel W3) and ozone-resistant (Bel B) tobacco cultivars as well as bean cv. Nerina and petunia cv. White cascade, both recognized as ozone sensitive, were used in the experiment. Investigations were carried out at two exposure sites varying in tropospheric ozone levels. Ozone negatively affected the leaf growth of both tobacco cultivars and bean. A negative relation was also found for ozone concentration and tobacco plant height. Number of petunia flowers and stalks and bean pods was positively correlated with ozone concentration. This could have been connected with earlier plant maturation due to faster generative development of plants in ozone-stress conditions.     

Ectopic Expression of the Pttkn1 Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm

A novel knottedl-like homeobox (knox) gene, Pttknl (Populus tremula×tremuloides knotted1), isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leafdisc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on theabaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petal color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed.     

Ectopic Expression of the Pttkn1 Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm

A novel knottedl-like homeobox (knox) gene, Pttknl (Populus tremula×tremuloides knotted1), isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leafdisc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on theabaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petal color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed.     

Organ boundary NAC‐domain transcription factors are implicated in the evolution of petal fusion

• Research rationale: Evolution of fused petals (sympetaly) is considered to be an important innovation that has repeatedly led to increased pollination efficiency, resulting in accelerated rates of plant diversification. Although little is known about the underlying regulation of sympetaly, genetic pathways ancestrally involved in organ boundary establishment (e.g. CUP SHAPED COTYLEDON [CUC] 1–3 genes) are strong candidates. In sympetalous petunia, mutations in the CUC1/2‐like orthologue NO APICAL MERISTEM (NAM) inhibit shoot apical meristem formation. Despite this, occasional ‘escape shoots’ develop flowers with extra petals and fused inter‐floral whorl organs.
• Central methods: To To determine if petunia CUC‐like genes regulate additional floral patterning, we used virus‐induced silencing (VIGS) following establishment of healthy shoot apices to re‐examine the role of NAM in petunia petal development, and uniquely characterise the CUC3 orthologue NH16.
• Key results: Confirming previous results, we found that reduced floral NAM/NH16 expression caused increased petal–stamen and stamen–carpel fusion, and often produced extra petals. However, further to previous results, all VIGS plants infected with NAM or NH16 constructs exhibited reduced fusion in the petal whorl compared to control plants.
• Main conclusions: Together with previous data, our results demonstrate conservation of petunia CUC‐like genes in establishing inter‐floral whorl organ boundaries, as well as functional evolution to affect the fusion of petunia petals.

     

Tissue-Dependent Expression of the Rice wx+ Gene Promoter in Transgenic Rice and Petunia

The waxy (wx) locus, which controls the amylose synthesis, isknown to be expressed specifically in the endosperm and pollen.To study the tissue-specific regulation of the wx⁺ gene, weintroduced a fusion gene that consisted of the upstream sequenceof the wx⁺ gene and the gene for rß-glucuronidase(GUS) into cells of rice (Oryza sativa L.) and petunia (Petuniahybrida L.). GUS activity was examined in the regenerated transgenicrice and petunia plants. In transgenic rice, the upstream sequenceof the wx⁺ gene was sufficient to direct the tissue-specificexpression of GUS in the endosperm and pollen, and the controlof expression was quantitative. By contrast, in transgenic petunia,the same fusion gene was expressed in pollen but not in theendosperm. These results suggest that the putative cis-actingelements that direct pollen-specific expression are common toor similar in both monocotyledonous and dicotyledonous plants,whereas ciy-elements responsible for the endosperm-specificexpression of the rice wx⁺ gene do not function in petunia,in which development of the endosperm differs from that in rice. ⁴Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan     

Overexpression of Xanthomonas campestris pv. vesicatoria effector AvrBsT in Arabidopsis triggers plant cell death, disease and defense responses

Recognition of bacterial effector proteins by plant cells is crucial for plant disease and defense response signaling. The Xanthomonas campestris pv. vesicatoria (Xcv) type III effector protein, AvrBsT, is secreted into plant cells from Xcv strain Bv5-4a. Here, we demonstrate that dexamethasone (DEX): avrBsT overexpression triggers cell death signaling in healthy transgenic Arabidopsis plants. AvrBsT overexpression in Arabidopsis also reduced susceptibility to infection with the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Overexpression of avrBsT significantly induced some defense-related genes in Arabidopsis leaves. A high-throughput in planta proteomics screen identified TCP-1 chaperonin, SEC7-like guanine nucleotide exchange protein and calmodulin-like protein, which were differentially expressed in DEX:avrBsT-overexpression (OX) Arabidopsis plants during Hp. arabidopsidis infection. Treatment with purified GST-tagged AvrBsT proteins distinctly inhibited the growth and sporulation of Hp. arabidopsidis on Arabdiopsis cotyledons. In contrast, DEX:avrBsT-OX plants exhibited enhanced susceptibility to Pseudomonas syringae pv. tomato (Pst) DC3000 infection. Notably, susceptible cell death and enhanced electrolyte leakage were significantly induced in the Pst-infected leaves of DEX:avrBsT-OX plants. Together, these results suggest that Xcv effector AvrBsT overexpression triggers plant cell death, disease and defense signaling leading to both disease and defense responses to microbial pathogens of different lifestyles.     

Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes

Glandular trichomes are the phytochemical factories of plants, and they secrete a wide range of commercially important natural products such as lipids, terpenes and flavonoids. Herein, we report that the Nicotiana tabacum LTP1 (NtLTP1) gene, which is specifically expressed in long glandular trichomes, plays a role in lipid secretion from trichome heads. NtLTP1 mRNA is abundantly transcribed in trichomes, but NtLTP3, NtLTP4 and NtLTP5 are not. In situ hybridization revealed that NtLTP1 mRNAs accumulate specifically in long trichomes and not in short trichomes or epidermal cells. X-gluc staining of leaves from a transgenic plant expressing the NtLTP1 promoter fused to a GUS gene revealed that NtLTP1 protein accumulated preferentially on the tops of long glandular trichomes. GFP fluorescence from transgenic tobacco plants expressing an NtLTP1-GFP fusion protein was localized at the periphery of cells and in the excreted liquid droplets from the glandular trichome heads. In vitro assays using a fluorescent 2-p-toluidinonaphthalene-6-sulfonate probe indicated that recombinant NtLTP1 had lipid-binding activity. The overexpression of NtLTP1 in transgenic tobacco plants resulted in the increased secretion of trichome exudates, including epicuticular wax. In transgenic NtLTP1-RNAi lines, liquid secretion from trichomes was strongly reduced, but epicuticular wax secretion was not altered. Moreover, transgenic tobacco plants overexpressing NtLTP1 showed increased protection against aphids. Taken together, these data suggest that NtLTP1 is abundantly expressed in long glandular trichomes, and may play a role in lipid secretion from long glandular trichomes.     

The bacterial effector DspA/E is toxic in Arabidopsis thaliana and is required for multiplication and survival of fire blight pathogen

The type III effector DspA/E is an essential pathogenicity factor of the phytopathogenic bacterium Erwinia amylovora. We showed that DspA/E was required for transient bacterial growth in nonhost Arabidopsis thaliana leaves, as an E. amylovora dspA/E mutant was unable to grow. We expressed DspA/E in A. thaliana transgenic plants under the control of an oestradiol‐inducible promoter, and found that DspA/E expressed in planta restored the growth of a dspA/E mutant. DspA/E expression in these transgenic plants led to the modulation by at least two‐fold of the expression of 384 genes, mostly induced (324 genes). Both induced and repressed genes contained high proportions of defence genes. DspA/E expression ultimately resulted in plant cell death without requiring a functional salicylic acid signalling pathway. Analysis of A. thaliana transgenic seedlings expressing a green fluorescent protein (GFP):DspA/E fusion indicated that the fusion protein could only be detected in a few cells per seedling, suggesting the degradation or absence of accumulation of DspA/E in plant cells. Consistently, we found that DspA/E repressed plant protein synthesis when injected by E. amylovora or when expressed in transgenic plants. Thus, we conclude that DspA/E is toxic to A. thaliana: it promotes modifications, among which the repression of protein synthesis could be determinant in the facilitation of necrosis and bacterial growth.     

The central role of PhEIN2 in ethylene responses throughout plant development in petunia

The plant hormone ethylene regulates many aspects of growth and development. Loss-of-function mutations in ETHYLENE INSENSITIVE2 (EIN2) result in ethylene insensitivity in Arabidopsis, indicating an essential role of EIN2 in ethylene signaling. However, little is known about the role of EIN2 in species other than Arabidopsis. To gain a better understanding of EIN2, a petunia (Petunia x hybrida cv Mitchell Diploid [MD]) homolog of the Arabidopsis EIN2 gene (PhEIN2) was isolated, and the role of PhEIN2 was analyzed in a wide range of plant responses to ethylene, many that do not occur in Arabidopsis. PhEIN2 mRNA was present at varying levels in tissues examined, and the PhEIN2 expression decreased after ethylene treatment in petals. These results indicate that expression of PhEIN2 mRNA is spatially and temporally regulated in petunia during plant development. Transgenic petunia plants with reduced PhEIN2 expression were compared to wild-type MD and ethylene-insensitive petunia plants expressing the Arabidopsis etr1-1 gene for several physiological processes. Both PhEIN2 and etr1-1 transgenic plants exhibited significant delays in flower senescence and fruit ripening, inhibited adventitious root and seedling root hair formation, premature death, and increased hypocotyl length in seedling ethylene response assays compared to MD. Moderate or strong levels of reduction in ethylene sensitivity were achieved with expression of both etr1-1 and PhEIN2 transgenes, as measured by downstream expression of PhEIL1. These results demonstrate that PhEIN2 mediates ethylene signals in a wide range of physiological processes and also indicate the central role of EIN2 in ethylene signal transduction.     

A petunia MADS box gene involved in the transition from vegetative to reproductive development.

We have identified a novel petunia MADS box gene, PETUNIA FLOWERING GENE (PFG), which is involved in the transition from vegetative to reproductive development. PFG is expressed in the entire plant except stamens, roots and seedlings. Highest expression levels of PFG are found in vegetative and inflorescence meristems. Inhibition of PFG expression in transgenic plants, using a cosuppression strategy, resulted in a unique nonflowering phenotype. Homozygous pfg cosuppression plants are blocked in the formation of inflorescences and maintain vegetative growth. In these mutants, the expression of both PFG and the MADS box gene FLORAL BINDING PROTEIN26 (FBP26), the putative petunia homolog of SQUAMOSA from Antirrhinum, are down-regulated. In hemizygous pfg cosuppression plants initially a few flowers are formed, after which the meristem reverts to the vegetative phase. This reverted phenotype suggests that PFG, besides being required for floral transition, is also required to maintain the reproductive identity after this transition. The position of PFG in the hierarchy of genes controlling floral meristem development was investigated using a double mutant of the floral meristem identity mutant aberrant leaf and flower (alf) and the pfg cosuppression mutant. This analysis revealed that the pfg cosuppression phenotype is epistatic to the alf mutant phenotype, indicating that PFG acts early in the transition to flowering. These results suggest that the petunia MADS box gene, PFG, functions as an inflorescence meristem identity gene required for the transition of the vegetative shoot apex to the reproductive phase and the maintenance of reproductive identity.     

Soybean AP1 homologs control flowering time and plant height

Flowering time and plant height are key agronomic traits that directly affect soybean (Glycine max) yield. APETALA1 (AP1) functions as a class A gene in the ABCE model for floral organ development, helping to specify carpel, stamen, petal, and sepal identities. There are four AP1 homologs in soybean, all of which are mainly expressed in the shoot apex. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR‐associated protein 9 technology to generate a homozygous quadruple mutant, gmap1, with loss‐of‐function mutations in all four GmAP1 genes. Under short‐day (SD) conditions, the gmap1 quadruple mutant exhibited delayed flowering, changes in flower morphology, and increased node number and internode length, resulting in plants that were taller than the wild type. Conversely, overexpression of GmAP1a resulted in early flowering and reduced plant height compared to the wild type under SD conditions. The gmap1 mutant and the overexpression lines also exhibited altered expression of several genes related to flowering and gibberellic acid metabolism, thereby providing insight into the role of GmAP1 in the regulatory networks controlling flowering time and plant height in soybean. Increased node number is the trait with the most promise for enhancing soybean pod number and grain yield. Therefore, the mutant alleles of the four AP1 homologs described here will be invaluable for molecular breeding of improved soybean yield.     

Calmodulin-dependent protein kinases phosphorylate gp130 at the serine-based dileucine internalization motif

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the low affinity LIF receptor (LIFR) and gp130. We previously demonstrated that LIF stimulation caused phosphorylation of gp130 at Ser782, adjacent to a dileucine internalization motif, and that transient expression of a mutant receptor lacking Ser782 resulted in increased cell surface expression and increased LIF-stimulated gene expression compared to wild-type receptor. Phosphorylation of Ser782 on gp130 fusion protein by LIF-stimulated 3T3-L1 cell extracts was inhibited 61% by autocamtide-2-related inhibitory peptide (AIP), a highly specific and highly effective inhibitor of calmodulin-dependent protein kinase type II (CaMKII). Purified rat forebrain CaMKII was also able to phosphorylate gp130 fusion protein at Ser782 in vitro. Furthermore, antibodies targeting CaMKII and CaMKIV were able to immunoprecipitate gp130 phosphorylating activity from LIF-stimulated 3T3-L1 lysates. While pretreatment of cells with the MAPKK inhibitors PD98059 and U0126 blocked phosphorylation of Ser782 prior to LIF stimulation, these inhibitors did not block Ser782 phosphorylation by LIF-stimulated 3T3-L1 cell extracts in vitro. These results show that CaMKII and possibly CaMKIV phosphorylate Ser782 in the serine-based dileucine internalization motif of gp130 via a MAPK-dependent pathway.