Nayelli Marsch-Martínez

Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene

Freshwater is a limited and dwindling global resource; therefore, efficient water use is required for food crops that have high water demands, such as rice, or for the production of sustainable energy biomass. We show here that expression of the Arabidopsis HARDY (HRD) gene in rice improves water use efficiency, the ratio of biomass produced to the water used, by enhancing photosynthetic assimilation and reducing transpiration. These drought-tolerant, low-water-consuming rice plants exhibit increased shoot biomass under well irrigated conditions and an adaptive increase in root biomass under drought stress. The HRD gene, an AP2/ERF-like transcription factor, identified by a gain-of-function Arabidopsis mutant hrd-D having roots with enhanced strength, branching, and cortical cells, exhibits drought resistance and salt tolerance, accompanied by an enhancement in the expression of abiotic stress associated genes. HRD overexpression in Arabidopsis produces thicker leaves with more chloroplast-bearing mesophyll cells, and in rice, there is an increase in leaf biomass and bundle sheath cells that probably contributes to the enhanced photosynthesis assimilation and efficiency. The results exemplify application of a gene identified from the model plant Arabidopsis for the improvement of water use efficiency coincident with drought resistance in the crop plant rice.

Activation Tagging Using the En-I Maize Transposon System in Arabidopsis

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays)En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant calledthread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that theEn-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.

Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs

Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies.

ASYMMETRIC LEAVES2-LIKE1gene, a member of the AS2/LOB family, controls proximal–distal patterning in Arabidopsis petals

The formation and the development of the floral organs require an intercalate expression of organ-specific genes. At the same time, meristem-specific genes are repressed to complete the differentiation of the organs in the floral whorls. In an Arabidopsis activation tagging population, a mutant affected in inflorescence architecture was identified. This gain-of-function mutant, designateddownwards siliques1 (dsl1-D), has shorter internodes and the lateral organs such as flowers are bending downwards, similar to the loss-of-function brevipedicellus (bp) mutant. The affected gene in dsl1-D appeared to be ASYMMETRIC LEAVES2-LIKE1 (ASL1)/LATERAL ORGAN BOUNDARIESdomain gene 36 (LBD36), which is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. Analysis of the loss-of-function mutant asl1/lbd36 did not show morphological aberration. Double mutant analysis of asl1/lbd36 together with as2, the ASL1/LBD36 closest homologue, demonstrates that these two members of the AS2/LOB family act partially redundant to control cell fate determination in Arabidopsis petals. Moreover, molecular analysis revealed that overexpression of ASL1/LBD36 leads to repression of the homeobox gene BP, which supports the model that an antagonistic relationship between ASL/LBD and homeobox members is required for the differentiation of lateral organs.

BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathways.

The BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number. Moreover, severe overexpressors showed ectopic callus formation in roots. Accordingly, global gene expression analysis using the overexpression mutant reflected the alterations in cell proliferation, differentiation and growth through expression changes in RBR, CYCD, and TCP genes, as well as genes involved in cell expansion (i.e. expansins and the actin remodeling factor ADF5). Furthermore, the expression of hormone signaling (i.e. auxin and cytokinin), biosynthesis (i.e. ethylene and jasmonic acid) and regulatory genes was found to be perturbed in bol-D mutant leaves.

The role of cytokinin during Arabidopsis gynoecia and fruit morphogenesis and patterning

Cytokinins have many essential roles in embryonic and post-embryonic growth and development, but their role in fruit morphogenesis is currently not really known. Moreover, information about the spatio-temporal localization pattern of cytokinin signaling in gynoecia and fruits is lacking. Therefore, the synthetic reporter line TCS::GFP was used to visualize cytokinin signaling during gynoecium and fruit development. Fluorescence was detected at medial regions of developing gynoecia, and, unexpectedly, at the valve margin in developing fruits, and was severely altered in mutants that lack or ectopically acquire valve margin identity. Comparison to developing gynoecia and fruits in a DR5rev::GFP line showed that the transcriptional responses to cytokinin and auxin are frequently present in complementary patterns. Moreover, cytokinin treatments in early gynoecia produced conspicuous changes, and treatment of valve margin mutant fruits restored this tissue. The results suggest that the phytohormone cytokinin is important in gynoecium and fruit patterning and morphogenesis, playing at least two roles: an early proliferation-inducing role at the medial tissues of the developing gynoecia, and a late role in fruit patterning and morphogenesis at the valve margin of developing fruits.

The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression

Elucidating molecular links between cell‐fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell‐fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem‐cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS‐box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem‐cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS‐domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4‐dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning.

Cytochrome P450 CYP78A9 Is Involved in Arabidopsis Reproductive Development

The cyp78a8 cyp78a9 loss-of-function mutant has reduced seed set due to outer integument development arrest leading to female sterility. Synchronized communication between gametophytic and sporophytic tissue is crucial for successful reproduction, and hormones seem to have a prominent role in it. Here, we studied the role of the Arabidopsis (Arabidopsis thaliana) cytochrome P450 CYP78A9 enzyme during reproductive development. First, controlled pollination experiments indicate that CYP78A9 responds to fertilization. Second, while CYP78A9 overexpression can uncouple fruit development from fertilization, the cyp78a8 cyp78a9 loss-of-function mutant has reduced seed set due to outer ovule integument development arrest, leading to female sterility. Moreover, CYP78A9 has a specific expression pattern in inner integuments in early steps of ovule development as well as in the funiculus, embryo, and integuments of developing seeds. CYP78A9 overexpression did not change the response to the known hormones involved in flower development and fruit set, and it did not seem to have much effect on the major known hormonal pathways. Furthermore, according to previous predictions, perturbations in the flavonol biosynthesis pathway were detected in cyp78a9, cyp78a8 cyp78a9, and empty siliques (es1-D) mutants. However, it appeared that they do not cause the observed phenotypes. In summary, these results add new insights into the role of CYP78A9 in plant reproduction and present, to our knowledge, the first characterization of metabolite differences between mutants in this gene family.

JAIBA, a class-II HD-ZIP transcription factor involved in the regulation of meristematic activity, and important for correct gynoecium and fruit development in Arabidopsis

The gynoecium is one of the most complex organs of a plant. After fertilization, it becomes a fruit, which has two important functions: to protect the seeds while they develop and to disperse them at maturity. The development and patterning of the gynoecium and later fruit must be finely regulated to ensure the survival of the species that produces them. The process that leads to successful fruit formation starts at early stages of floral meristem development and follows a series of chronologically successive events. In this work we report the functional characterization of the class-II homeodomain leucine zipper (HD-ZIP) JAIBA (JAB) gene. Mutant jab plants show sporophytic defects in male and female reproductive development, and combined with crabs claw cause defects in the floral meristem (FM) determination process and gynoecium medial tissue development. This suggests that proper FM determination is required for gynoecium medial tissue development, and indicates that JAB and CRC are necessary for both processes. Furthermore, the JAB protein interacts with transcription factors known to regulate meristematic activity, fruit development, and floral meristem determinacy. The sporophytic effect on pollen and embryo sac development might be an independent and later function of JAB. In summary, we present data that indicates that the JAB gene regulates meristematic activity in different tissues, and that it is necessary for the correct formation of the gynoecium at different stages, contributing to a crucial process in plant life: proper fruit development.

Cytokinin treatments affect the apical-basal patterning of the Arabidopsis gynoecium and resemble the effects of polar auxin transport inhibition

The apical-basal axis of the Arabidopsis gynoecium is established early during development and is divided into four elements from the bottom to the top: the gynophore, the ovary, the style, and the stigma. Currently, it is proposed that the hormone auxin plays a critical role in the correct apical-basal patterning through a concentration gradient from the apical to the basal part of the gynoecium, as chemical inhibition of polar auxin transport through 1-N-naphtylphtalamic acid (NPA) application, severely affects the apical-basal patterning of the gynoecium. In this work, we show that the apical-basal patterning of gynoecia is also sensitive to exogenous cytokinin (benzyl amino purine, BAP) application in a similar way as to NPA. BAP and NPA treatments were performed in different mutant backgrounds where either cytokinin perception or auxin transport and perception were affected. We observed that cytokinin and auxin signaling mutants are hypersensitive to NPA treatment, and auxin transport and signaling mutants are hypersensitive to BAP treatment. BAP effects in apical-basal gynoecium patterning are very similar to the effects of NPA, therefore, it is possible that BAP affects auxin transport in the gynoecium. Indeed, not only the cytokinin-response TCS::GFP marker, but also the auxin efflux carrier PIN1 (PIN1::PIN1:GFP) were both affected in BAP-induced valveless gynoecia, suggesting that the BAP treatment producing the morphological changes has an impact on both in the response pattern to cytokinin and on auxin transport. In summary, we show that cytokinin affects proper apical-basal gynoecium patterning in Arabidopsis in a similar way to the inhibition of polar auxin transport, and that auxin and cytokinin mutants and markers suggest a relation between both hormones in this process.