Genetic Components of Root Architecture Remodeling in Response to Salt Stress.

Salinity of the soil is highly detrimental to plant growth. Plants respond by a redistribution of root mass between main and lateral roots, yet the genetic machinery underlying this process is still largely unknown. Here, we describe the natural variation among 347 Arabidopsis thaliana accessions in root system architecture (RSA) and identify the traits with highest natural variation in their response to salt. Salt-induced changes in RSA were associated with 100 genetic loci using genome-wide association studies. Two candidate loci associated with lateral root development were validated and further investigated. Changes in CYP79B2 expression in salt stress positively correlated with lateral root development in accessions, and cyp79b2 cyp79b3 double mutants developed fewer and shorter lateral roots under salt stress, but not in control conditions. By contrast, high HKT1 expression in the root repressed lateral root development, which could be partially rescued by addition of potassium. The collected data and multivariate analysis of multiple RSA traits, available through the Salt_NV_Root App, capture root responses to salinity. Together, our results provide a better understanding of effective RSA remodeling responses, and the genetic components involved, for plant performance in stress conditions.

Capturing Arabidopsis root architecture dynamics with ROOT-FIT reveals diversity in responses to salinity.

The plant root is the first organ to encounter salinity stress, but the effect of salinity on root system architecture (RSA) remains elusive. Both the reduction in main root (MR) elongation and the redistribution of the root mass between MRs and lateral roots (LRs) are likely to play crucial roles in water extraction efficiency and ion exclusion. To establish which RSA parameters are responsive to salt stress, we performed a detailed time course experiment in which Arabidopsis (Arabidopsis thaliana) seedlings were grown on agar plates under different salt stress conditions. We captured RSA dynamics with quadratic growth functions (root-fit) and summarized the salt-induced differences in RSA dynamics in three growth parameters: MR elongation, average LR elongation, and increase in number of LRs. In the ecotype Columbia-0 accession of Arabidopsis, salt stress affected MR elongation more severely than LR elongation and an increase in LRs, leading to a significantly altered RSA. By quantifying RSA dynamics of 31 different Arabidopsis accessions in control and mild salt stress conditions, different strategies for regulation of MR and LR meristems and root branching were revealed. Different RSA strategies partially correlated with natural variation in abscisic acid sensitivity and different Na(+)/K(+) ratios in shoots of seedlings grown under mild salt stress. Applying root-fit to describe the dynamics of RSA allowed us to uncover the natural diversity in root morphology and cluster it into four response types that otherwise would have been overlooked.

Role of Na+, K+, Cl-, proline and sucrose concentrations in determining salinity tolerance and their correlation with the expression of multiple genes in tomato.

One of the major abiotic stresses affecting agriculture is soil salinity, which reduces crop yield and, consequently, revenue for farmers. Although tomato is an important agricultural species, elite varieties are poor at withstanding salinity stress. Thus, a feasible way of improving yield under conditions of salinity stress is to breed for improved salt tolerance. In this study, we analysed the physiological and genetic parameters of 23 tomato accessions in order to identify possible traits to be used by plant breeders to develop more tolerant tomato varieties. Although we observed a wide range of Na(+) concentrations within the leaves, stems and roots, the maintenance of growth in the presence of 100 mM NaCl did not correlate with the exclusion or accumulation of Na(+). Nor could we correlate the growth with accumulation of sugars and proline or with the expression of any gene involved in the homoeostasis of Na(+) in the plant. However, several significant correlations between gene expression and Na(+) accumulation were observed. For instance, Na(+) concentrations both in the leaves and stems were positively correlated with HKT1;2 expression in the roots, and Na(+) concentration measured in the roots was positively correlated with HKT1;1 expression also in the roots. Higher and lower Na(+) accumulation in the roots and leaves were significantly correlated with higher NHX3 and NHX1 expression in the roots, respectively. These results suggest that, in tomato, for a particular level of tolerance to salinity, a complex relationship between Na(+) concentration in the cells and tissue tolerance defines the salinity tolerance of individual tomato accessions. In tomato it is likely that tissue and salinity tolerance work independently, making tolerance to salinity depend on their relative effects rather than on one of these mechanisms alone.

Identification of a resistance gene Rpi-dlc1 to Phytophthora infestans in European accessions of Solanum dulcamara.

Initial screening of 14 Solanum dulcamara accessions enabled the identification of individuals resistant and susceptible to Phytophthora infestans. Crosses between contrasting genotypes resulted in three F(2)-BC(1) populations segregating for resistance to late blight in a laboratory assay and under field conditions. Genetic profiling of one of these populations using 128 AFLP primers generated three markers linked to the resistant phenotype. Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers. Thus, we describe a first resistant gene, named Rpi-dlc1, from S. dulcamara, a Solanum species native to Europe. In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence. This indicated the possible presence of additional Rpi genes.

Abscisic acid levels in tomato ovaries are regulated by LeNCED1 and SlCYP707A1.

Although the hormones, gibberellin and auxin, are known to play a role in the initiation of fruits, no such function has yet been demonstrated for abscisic acid (ABA). However, ABA signaling and ABA responses are high in tomato (Solanum lycopersicum L.) ovaries before pollination and decrease thereafter (Vriezen et al. in New Phytol 177:60-76, 2008). As a first step to understanding the role of ABA in ovary development and fruit set in tomato, we analyzed ABA content and the expression of genes involved in its metabolism in relation to pollination. We show that ABA levels are relatively high in mature ovaries and decrease directly after pollination, while an increase in the ABA metabolite dihydrophaseic acid was measured. An important regulator of ABA biosynthesis in tomato is 9-cis-epoxy-carotenoid dioxygenase (LeNCED1), whose mRNA level in ovaries is reduced after pollination. The increased catabolism is likely caused by strong induction of one of four newly identified putative (+)ABA 8'-hydroxylase genes. This gene was named SlCYP707A1 and is expressed specifically in ovules and placenta. Transgenic plants, overexpressing SlCYP707A1, have reduced ABA levels and exhibit ABA-deficient phenotypes suggesting that this gene encodes a functional ABA 8'-hydroxylase. Gibberellin and auxin application have different effects on the LeNCED1 and SlCYP707A1 gene expression. The crosstalk between auxins, gibberellins and ABA during fruit set is discussed.

The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development.

Auxin response factors (ARFs) are encoded by a gene family of transcription factors that specifically control auxin-dependent developmental processes. A tomato ARF gene, homologous to Arabidopsis NPH4/ARF7 and therefore designated as Solanum lycopersicum ARF7 (SlARF7), was found to be expressed at a high level in unpollinated mature ovaries. More detailed analysis of tomato ovaries showed that the level of SlARF7 transcript increases during flower development, remains at a constant high level in mature flowers, and is down-regulated within 48 h after pollination. Transgenic plants with decreased SlARF7 mRNA levels formed seedless (parthenocarpic) fruits. These fruits were heart-shaped and had a rather thick pericarp due to increased cell expansion, compared with the pericarp of wild-type fruits. The expression analysis, together with the parthenocarpic fruit phenotype of the transgenic lines, suggests that, in tomato, SlARF7 acts as a negative regulator of fruit set until pollination and fertilization have taken place, and moderates the auxin response during fruit growth.

Changes in tomato ovary transcriptome demonstrate complex hormonal regulation of fruit set.

Plant hormones are considered to be important mediators of the fruit developmental signal after pollination. The role of phytohormones in tomato (Solanum lycopersicum) fruit set was investigated here. Transcriptome analysis of ovaries was performed using two complementary approaches: cDNA-amplified fragment length polymorphism (AFLP) and microarray analysis. The gene expression profiles obtained suggest that, in addition to auxin and gibberellin, ethylene and abscisic acid (ABA) are involved in regulating fruit set. Before fruit development, many genes involved in biotic and abiotic responses are active in the ovary. In addition, genes involved in ethylene and ABA biosynthesis were strongly expressed, suggesting relatively high ethylene and ABA concentrations before fruit set. Induction of fruit development, either by pollination or by gibberellin application, attenuated expression of all ethylene and ABA biosynthesis and response genes within 24 h. It is proposed that the function of ABA and ethylene in fruit set might be antagonistic to that of auxin and gibberellin in order to keep the ovary in a temporally protected and dormant state; either to protect the ovary tissue or to prevent fruit development before pollination and fertilization occur.

Expression and localization of calreticulin in tobacco anthers and pollen tubes.

The developmental expression pattern and localization of calreticulin were studied in Nicotiana tabacum L. anthers, pollen and pollen tubes. High transcript and protein levels were detected throughout anther development. Immunolocalization of calreticulin in the anthers showed particular dense label in tapetum and pollen at developmental stage 2, when the tapetum is highly active and the pollen tetrads are formed. Much lower transcript and protein levels were detected in dry and hydrated pollen and in pollen tubes. Immunofluorescence labeling of both chemically fixed and cryo-fixed and freeze-substituted pollen tubes showed the presence of calreticulin in Golgi apparatus and endoplasmic reticulum (ER). Calreticulin was seen throughout the stacks in the Golgi apparatus and in the areas with coated-Golgi vesicles but much less so in the ER. Calreticulin was not found in the secretory vesicles. A relatively intense label was occasionally seen adjacent to the wall of the tube. No significant label was observed in mitochondria, vacuoles, generative cells, cell wall or callose plugs. The present results are consistent with a role of calreticulin in Ca2+-dependent folding of secreted glycoproteins in tapetum, pollen and pollen tubes.

Lipid transfer proteins enhance cell wall extension in tobacco.

Plant cells are enclosed by a rigid cell wall that counteracts the internal osmotic pressure of the vacuole and limits the rate and direction of cell enlargement. When developmental or physiological cues induce cell extension, plant cells increase wall plasticity by a process called loosening. It was demonstrated previously that a class of proteins known as expansins are mediators of wall loosening. Here, we report a type of cell wall-loosening protein that does not share any homology with expansins but is a member of the lipid transfer proteins (LTPs). LTPs are known to bind a large range of lipid molecules to their hydrophobic cavity, and we show here that this cavity is essential for the cell wall-loosening activity of LTP. Furthermore, we show that LTP-enhanced wall extension can be described by a logarithmic time function. We hypothesize that LTP associates with hydrophobic wall compounds, causing nonhydrolytic disruption of the cell wall and subsequently facilitating wall extension.

STIG1 controls exudate secretion in the pistil of petunia and tobacco.

The lipid-rich, sticky exudate covering the stigma of solanaceous species such as tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) contains several proteins, of which only some have been characterized to date. Proteome analysis of the stigmatic exudate in both species revealed the presence of a cysteine-rich, slightly acidic 12-kD protein called stigma-specific protein 1 (STIG1). In both tobacco and petunia, Stig1 is highly expressed at the mRNA level in very young and developing flowers, whereas hardly any Stig1 transcript is detected in mature flowers. This expression pattern coincides with the differentiation of the secretory zone, forming the intercellular spaces into which the exudate is secreted. Using reverse genetics, we show that STIG1 is involved in the secretion and merging of exudate lipids in the intercellular spaces of the secretory zone and that plants lacking STIG1 show an accelerated deposition of exudate onto the stigmatic surface. This phenotype was observed both in a petunia knockout mutant and in tobacco transgenic plants. We therefore propose that STIG1 plays a role in the temporal regulation of the essential exudate secretion onto the stigma.

PIP1 and PIP2 aquaporins are differentially expressed during tobacco anther and stigma development.

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues, such as occurs during dehiscence of the anther and hydration of the pollen grain after it is deposited on a stigma. To get more insight in these processes, a set of putative aquaporins was cloned and it was found that at least 15 are expressed in reproductive organs, which indicates that the control of water flow is important for reproduction. Functional studies in Xenopus laevis oocytes using two of the cDNAs showed that NtPIP2;1 is an efficient aquaporin, whereas NtPIP1;1 is not. Expression studies on RNA and protein levels showed that PIP1 and PIP2 genes are differently expressed in reproductive organs: PIP1 RNA accumulates in the stigma, and PIP1 and PIP2 RNA can be detected in most tissues of the anther.

Pollination modulates expression of the PPAL gene, a pistil-specific beta-expansin.

Using differential screening we isolated a pistil-specific cDNA clone corresponding to a 1.2 kb mRNA and encoding a 32.5 kDa protein. The amino acid sequence shared similarity with that of group-I grass pollen allergens, which are known to have expansin activity. This clone, which later showed to share homology also with beta-expansins, was named PPAL. The PPAL mRNA was specifically expressed in the secretory zone of the stigma and in the epidermal layer of the placenta. The accumulation level of the transcript increased during pollination, and the protein was secreted in the stigmatic exudate of the tobacco flower. We suggest here that PPAL is a new expansin, acting as a cell-wall-loosening agent during pollination.