The tapetal tissue is essential for the maintenance of redox homeostasis during microgametogenesis in tomato.

The tapetum is a specialized layer of cells within the anther, adjacent to the sporogenous tissue. During its short life, it provides nutrients, molecules and materials to the pollen mother cells and microsporocytes, being essential during callose degradation and pollen wall formation. The interaction between the tapetum and sporogenous cells in Solanum lycopersicum (tomato) plants, despite its importance for breeding purposes, is poorly understood. To investigate this process, gene editing was used to generate loss-of-function mutants that showed the complete and specific absence of tapetal cells. These plants were obtained targeting the previously uncharacterized Solyc03g097530 (SlTPD1) gene, essential for tapetum specification in tomato plants. In the absence of tapetum, sporogenous cells developed and callose deposition was observed. However, sporocytes failed to undergo the process of meiosis and finally degenerated, leading to male sterility. Transcriptomic analysis conducted in mutant anthers lacking tapetum revealed the downregulation of a set of genes related to redox homeostasis. Indeed, mutant anthers showed a reduction in the accumulation of reactive oxygen species (ROS) at early stages and altered activity of ROS-scavenging enzymes. The results obtained highlight the importance of the tapetal tissue in maintaining redox homeostasis during male gametogenesis in tomato plants.

Production of engineered long-life and male sterile Pelargonium plants.

BACKGROUND: Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. RESULTS: The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3-4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. CONCLUSION: The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.

Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation.

Engineered male sterility in ornamental plants has many applications such as facilitate hybrid seed production, eliminate pollen allergens, reduce the need for deadheading to extend the flowering period, redirect resources from seeds to vegetative growth, increase flower longevity and prevent gene flow between genetically modified and related native plants. We have developed a reliable and efficient Agrobacterium-mediated protocol for the genetic transformation of different Kalanchoe blossfeldiana commercial cultivars. Transformation efficiency for cv. 'Hillary' was 55.3% whereas that of cv. 'Tenorio' reached 75.8%. Selection was carried out with the nptII gene and increasing the kanamycin concentration from 25 to 100 mg l(-1) allowed to reduced escapes from 50 to 60% to virtually 0%. This method was used to produce male-sterile plants through engineered anther ablation. In our approach, we tested a male sterility chimaeric gene construct (PsEND1::barnase) to evaluate its effectiveness and effect on phenotype. No significant differences were found in the growth patterns between the transgenic lines and the wild-type plants. No viable pollen grains were observed in the ablated anthers of any of the lines carrying the PsEND1::barnase construct, indicating that the male sterility was complete. In addition, seed set was completely abolished in all the transgenic plants obtained. Our engineered male-sterile approach could be used, alone or in combination with a female-sterility system, to reduce the invasive potential of new ornamentals, which has become an important environmental problem in many countries.

Analysis of B function in legumes: PISTILLATA proteins do not require the PI motif for floral organ development in Medicago truncatula.

The B-class gene PISTILLATA (PI) codes for a MADS-box transcription factor required for floral organ identity in angiosperms. Unlike Arabidopsis, it has been suggested that legume PI genes contribute to a variety of processes, such as the development of floral organs, floral common petal-stamen primordia, complex leaves and N-fixing root nodules. Another interesting feature of legume PI homologues is that some of them lack the highly conserved C-terminal PI motif suggested to be crucial for function. Therefore, legume PI genes are useful for addressing controversial questions on the evolution of B-class gene function, including how they may have diverged in both function and structure to affect different developmental processes. However, functional analysis of legume PI genes has been hampered because no mutation in any B-class gene has been identified in legumes. Here we fill this gap by studying the PI function in the model legume species Medicago truncatula using mutant and RNAi approaches. Like other legume species, M. truncatula has two PI homologues. The expression of the two genes, MtPI and MtNGL9, has strongly diverged, suggesting differences in function. Our analyses show that these genes are required for petal and stamen identity, where MtPI appears to play a predominant role. However, they appear not to be required for development of the nodule, the common primordia or the complex leaf. Moreover, both M. truncatula PI homologues lack the PI motif, which indicates that the C-terminal motif is not essential for PI activity.

Androesterilidad inducida mediante ingeniería genética en plantas: fundamentos y aplicaciones biotecnológicas / Male-sterility obtained by genetic engineering: principles and biotechnological applications

La disponibilidad de genotipos de plantas androestériles es crucial para laobtención de semillas híbridas y abre la posibilidad del manejo de las plantas deforma más respetuosa con el medio ambiente. Nosotros hemos desarrollado herramientasbiotecnológicas para la producción de plantas androestériles de interésagronómico (tomate, colza, tabaco) mediante el uso de la región promotora del genPsEND1 de guisante para dirigir la expresión de agentes citotóxicos específicamentea los tejidos estructurales de las anteras para producir su ablación genética. Enlas plantas androestériles obtenidas mediante ingeniería genética, hemos observadoque se produce un mayor número de ramas y consecuentemente una mayorproducción de flores. Además, la vida útil de estas plantas se prolonga de formanotable. Estas características son de interés para el sector de la floricultura, ya queactualmente se están produciendo híbridos mediante mejora convencional conflores muy vistosas y colores novedosos, pero con escasa producción de flores por planta. Por otra parte, la introducción en la planta modificada genéticamente deun gen que le confiera androesterilidad, es otra característica deseable en el campode las plantas ornamentales ya que evitaría la transferencia horizontal de transgenesal medio ambiente y a especies sexualmente compatibles

The availability of male-sterile plant varieties is relevant for obtaining of hybridplant lines which are more vigorous than the corresponding parental pure linesbecause the phenomenon known as heterosis. Moreover, the use of male-sterileplants prevents undesirable horizontal gene transfer. We have developed biotechnologicaltools to obtain androsteryle lines of plants with agronomic interest suchas tomato, tobacco, rape seed and wheat. This is accomplished by the use of thepromoter region of the PsEND1 gene to drive the expression of cytotoxic agents,such as barnase, to the structural tissues of the anthers. The male-sterile transgenicplants obtained live longer and show a higher number of branches and flowersthan the corresponding wild type plants. This will allow plant breeders to incorporatethose valuable characteristics to increase the number of flowers of plantsalready displaying new colours, shapes and fragrances. These new ornamentalplants are environmental friendly since horizontal gene transfer can not take place

Isolation of mtpim proves Tnt1 a useful reverse genetics tool in Medicago truncatula and uncovers new aspects of AP1-like functions in legumes.

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.

Functional conservation of PISTILLATA activity in a pea homolog lacking the PI motif.

Current understanding of floral development is mainly based on what we know from Arabidopsis (Arabidopsis thaliana) and Antirrhinum majus. However, we can learn more by comparing developmental mechanisms that may explain morphological differences between species. A good example comes from the analysis of genes controlling flower development in pea (Pisum sativum), a plant with more complex leaves and inflorescences than Arabidopsis and Antirrhinum, and a different floral ontogeny. The analysis of UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), the pea orthologs of LEAFY and UNUSUAL FLORAL ORGANS, has revealed a common link in the regulation of flower and leaf development not apparent in Arabidopsis. While the Arabidopsis genes mainly behave as key regulators of flower development, where they control the expression of B-function genes, UNI and STP also contribute to the development of the pea compound leaf. Here, we describe the characterization of P. sativum PISTILLATA (PsPI), a pea MADS-box gene homologous to B-function genes like PI and GLOBOSA (GLO), from Arabidopsis and Antirrhinum, respectively. PsPI encodes for an atypical PI-type polypeptide that lacks the highly conserved C-terminal PI motif. Nevertheless, constitutive expression of PsPI in tobacco (Nicotiana tabacum) and Arabidopsis shows that it can specifically replace the function of PI, being able to complement the strong pi-1 mutant. Accordingly, PsPI expression in pea flowers, which is dependent on STP, is identical to PI and GLO. Interestingly, PsPI is also transiently expressed in young leaves, suggesting a role of PsPI in pea leaf development, a possibility that fits with the established role of UNI and STP in the control of this process.