Simonetta Santi

Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots

Here, we have analysed the H(+)-ATPase-mediated extrusion of protons across the plasma membrane (PM) of rhizodermic cells, a process that is inducible by iron (Fe) deficiency and thought to serve in the mobilization of sparingly soluble Fe sources. The induction and function of Fe-responsive PM H(+)-ATPases in Arabidopsis roots was investigated by gene expression analysis and by using mutants defective in the expression or function of one of the isogenes. In addition, the expression of the most responsive isogenes was investigated in natural Arabidopsis accessions that have been selected for their in vivo proton extrusion activity. Our data suggest that the rhizosphere acidification in response to Fe deficiency is chiefly mediated by AHA2, while AHA1 functions as a housekeeping isoform. The aha7 knock-out mutant plants showed a reduced frequency of root hairs, suggesting an involvement of AHA7 in the differentiation of rhizodermic cells. Acidification capacity varied among Arabidopsis accessions and was associated with a high induction of AHA2 and IRT1, a high relative growth rate and a shoot-root ratio that was unaffected by the external Fe supply. An effective regulation of the Fe-responsive genes and a stable shoot-root ratio may represent important characteristics for the Fe uptake efficiency.

Water-extractable humic substances enhance iron deficiency responses by Fe-deficient cucumber plants

The ability of Fe-deficient cucumber plants to use iron complexed to a water-extractable humic substances fraction (WEHS), was investigated. Seven-day-old Fe-deficient plants were transferred to a nutrient solution supplemented daily for 5 days with 0.2 μM Fe as Fe-WEHS (5 μg org. C mL-1), Fe-EDTA, Fe-citrate or FeCl3. These treatments all allowed re-greening of the leaf tissue, and partial recovery of dry matter accumulation, chlorophyll and iron contents. However, the recovery was faster in plants supplied with Fe-WEHS and was already evident 48 h after Fe supply. The addition of 0.2 μM Fe to the nutrient solution caused also a partial recovery of the dry matter and iron accumulation in roots of Fe-deficient cucumber plants, particularly in those supplied with Fe-WEHS. The addition of WEHS alone (5 μg org. C mL-1, 0.04 μM Fe) to the nutrient solution slightly but significantly increased iron and chlorophyll contents in leaves of Fe-deficient plants; in these plants, dry matter accumulation in leaves and roots was comparable or even higher than that measured in plants treated with Fe-citrate or FeCl3. After addition of the different iron sources for 5 days to Fe-deficient roots, morphological modifications (proliferation of lateral roots, increase in the diameter of the sub-apical zones and amplified root-hair formation) and physiological responses (enhanced Fe(III)-chelate reductase and acidification of the nutrient solution) induced by Fe deficiency, were still evident, particularly in plants treated with the humic molecules. The presence of WEHS caused also a further acidification of the nutrient medium by Fe-deficient plants. The Fe-WEHS complex (1 μM Fe) could be reduced by intact cucumber roots, at rates of reduction higher than those measured for Fe-EDTA at equimolar iron concentration. Plasma membrane vesicles, purified by two-phase partition from root microsomes of Fe-deficient plants, were also able to reduce Fe-WEHS. Results show that Fe-deficient cucumber plants can use iron complexed to water soluble humic substances, at least in part via reduction of complexed Fe(III) by the plasma membrane Fe(III)-chelate reductase of root cells. In addition, the stimulating effect of humic substances on H+ release might be of relevance for the overall response of the plants to iron shortage.

Laser microdissection-assisted analysis of the functional fate of iron deficiency-induced root hairs in cucumber

Iron ranks fourth in the sequence of abundance of the elements in the Earths crust, but its low bio-availability often limits plant growth. When present in suboptimal amounts, the acquisition of iron by plants is aided by a suite of responses, comprising molecular and developmental changes that facilitate the uptake of iron from sparingly soluble pools. The expression of genes involved in the mobilization of iron (CsHA1), the reduction of ferric chelates (CsFRO1), and in the uptake of ferrous iron (CsIRT1) was investigated in epidermal cells of Fe-sufficient and Fe-deficient cucumber (Cucumis sativum L.) roots using the Laser Microdissection and Pressure Catapulting (LMPC) method. Growing plants hydroponically in media deprived of iron induced the differentiation of almost all epidermal cells into root hairs. No root hairs were formed under iron-replete conditions. The formation of root hairs in response to Fe starvation was associated with a dramatic increase in message levels of CsFRO1, CsIRT1, and the iron-inducible H(+)-ATPase isoform CsHA1, when compared to epidermal cells of Fe-sufficient plants. On the contrary, transcripts of a housekeeping ATPase isoform, CsHA2, were not detected in root hairs, suggesting that Fe-deficiency-induced acidification is predominantly mediated by CsHA1. These data show that the formation of root hairs in response to iron deficiency is associated with cell-specific accumulation of transcripts that are involved in iron acquisition. The results also show that this includes the differential regulation of ATPase isoforms with similar function, but supposedly different characteristics, to counteract the imbalance in nutrient supply efficiently.

Mapping gene activity of Arabidopsis root hairs

BackgroundQuantitative information on gene activity at single cell-type resolution is essential for the understanding of how cells work and interact. Root hairs, or trichoblasts, tubular-shaped outgrowths of specialized cells in the epidermis, represent an ideal model for cell fate acquisition and differentiation in plants.ResultsHere, we provide an atlas of gene and protein expression in Arabidopsis root hair cells, generated by paired-end RNA sequencing and LC/MS-MS analysis of protoplasts from plants containing a pEXP7-GFP reporter construct. In total, transcripts of 23,034 genes were detected in root hairs. High-resolution proteome analysis led to the reliable identification of 2,447 proteins, 129 of which were differentially expressed between root hairs and non-root hair tissue. Dissection of pre-mRNA splicing patterns showed that all types of alternative splicing were cell type-dependent, and less complex in EXP7-expressing cells when compared to non-root hair cells. Intron retention was repressed in several transcripts functionally related to root hair morphogenesis, indicative of a cell type-specific control of gene expression by alternative splicing of pre-mRNA. Concordance between mRNA and protein expression was generally high, but in many cases mRNA expression was not predictive for protein abundance.ConclusionsThe integrated analysis shows that gene activity in root hairs is dictated by orchestrated, multilayered regulatory mechanisms that allow for a cell type-specific composition of functional components.

Water-extractable humic substances alter root development and epidermal cell pattern in Arabidopsis

The effect of a low-molecular weight, water-extractable fraction of humic substances (WEHS) derived from sphagnum peat on post-embryonic plant development has been studied using Arabidopsis roots. Application of humic substances caused an array of changes in root morphology, such as an increase in root hair length and density, formation of ectopic root hairs, and an increase in cell proliferation in the root ground tissue. Application of WEHS affected genes involved in epidermal cell fate specification, suggesting that humic substances can alter developmental programs at an early stage of root cell differentiation. The WEREWOLF and GLABRA2 genes, encoding negative regulators of the root hair cell fate, were significantly down-regulated in the presence of WEHS. Thus, the presence of humic substances caused an ordered remodeling of the root morphology, leading to an increased absorptive surface of the root. Growth in the presence of WEHS did not rescue the phenotype of the root hair defective rhd6 mutant. Analyzing BA3:uidA and DR5:uidA transgenic plants, carrying auxin response elements, and monitoring the expression of the auxin-responsive GH3 gene by real-time RT-PCR did not provide evidence for a WEHS-induced expression of auxin-related genes. It is concluded that WEHS do not exert their effects in an auxin-like manner.

Soil humic substances stimulate proton release by intact oat seedling roots

Abstract The effect of a soil humic fraction (HS) on proton extrusion into deionized water by intact oat seedling roots was studied. In the presence of HS, at concentration of 10 μg organic carbon (C) mL‐1, a clear stimulation of acidification of the outer medium by the roots was observed after three to four hours of incubation. The addition of 0.5 mM vanadate to the solution bathing the roots drastically reduced the net proton extrusion, either in the presence or absence of HS, suggesting the involvement of the plasma membrane H+‐ATPase in the stimulation of the acidification of the outer medium by oat roots. The release of potassium (K) from the roots into deionized water was also monitored concomitantly to the proton extrusion. The loss of endogenous K from the roots was similar in the presence or absence of HS, while the recovery of the cation was slower in the presence of the humic fraction. However, after reabsorption of the released K, no net acidification was observed in control roots, while HS‐tr...

Laser microdissection of grapevine leaf phloem infected by stolbur reveals site-specific gene responses associated to sucrose transport and metabolism

Bois Noir is an emergent disease of grapevine that has been associated to a phytoplasma belonging to the XII-A stolbur group. In plants, phytoplasmas have been found mainly in phloem sieve elements, from where they spread moving through the pores of plates, accumulating especially in source leaves. To examine the expression of grapevine genes involved in sucrose transport and metabolism, phloem tissue, including sieve element/companion cell complexes and some parenchyma cells, was isolated from healthy and infected leaves by means of laser microdissection pressure catapulting (LMPC). Site-specific expression analysis dramatically increased sensitivity, allowing us to identify specific process components almost completely masked in whole-leaf analysis. Our findings showed decreased phloem loading through inhibition of sucrose transport and increased sucrose cleavage activity, which are metabolic changes strongly suggesting the establishment of a phytoplasma-induced switch from carbohydrate source to sink. The analysis focused at the infection site also showed a differential regulation and specificity of two pathogenesis-related thaumatin-like genes (TL4 and TL5) of the PR-5 family.

Recovery from stolbur disease in grapevine involves changes in sugar transport and metabolism

Grapevine can be severely affected by phytoplasmas, which are phytopathogenic Mollicutes invading the sieve elements of the host plant. The biochemical and molecular relationships between phytoplasmas and their hosts remain largely unexplored. Equally unknown is an interesting aspect of the pathogen–plant interaction called “recovery,” which is a spontaneous remission of symptoms in previously symptomatic plants. Recovered plants develop resistance mechanisms correlated with ultrastructural and biochemical changes in the sieve elements. Callose as well as sugars are involved in several plant defense processes and signaling. In the present work we have examined the possible involvement of callose, as well as callose synthase, sugar transporter, and cell wall invertase genes, during the infection and after “recovery” of grapevine from bois noir (BN). Ultrastructural investigation of leaf tissue showed that callose accumulated in the sieve elements of diseased grapevine; moreover, two genes encoding for callose synthase were up-regulated in the infected leaves. Regarding sucrose, expression analysis showed that sucrose transport and cleavage were severely affected by BN phytoplasma, which induced the establishment of a carbohydrate sink in the source leaf, and was analogous to other obligate biotrophs that acquire most of their nutrients from the host plant. Interestingly, whereas in recovered plants the transcript level of sucrose synthase was similar to healthy plants, sucrose transporters as well as cell wall invertase were expressed to a greater degree in recovered leaves than in healthy ones. Recovered plants seem to acquire structural and molecular changes leading to increases in sucrose transport ability and defense signaling.

Differentially-regulated defence genes in Malus domestica during phytoplasma infection and recovery

To improve knowledge about plant/phytoplasma interactions and, in particular, about the ‘recovery’ phenomenon in previously-infected plants, we investigated and compared expression levels of several defence-related genes (four pathogenesis-related proteins and three jasmonate-pathway marker enzymes) in apple plants showing different states of health: vigorous (healthy), phytoplasma-infected, and recovered. Real Time-PCR analyses demonstrated that genes are differentially expressed in apple leaf tissue according to the plants’ state of health. Malus domestica Pathogenesis-Related protein (MdPR) 1, MdPR 2 and MdPR 5 were significantly induced in leaves of diseased and symptomatic plants compared to leaves of those plants that were healthy or recovered. On the other hand, levels of all the jasmonate (JA)-pathway marker genes that we selected for this study, were up-regulated in the leaves of recovered plants compared to the diseased ones. In conclusion, our study demonstrated that two different sets of defence genes are involved in the interactions between apple plants and ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) and that these genes are differentially expressed during phytoplasma infection or recovery.

Sucrose transport and phloem unloading in peach fruit: potential role of two transporters localized in different cell types

Several complex physiological processes, which include long-distance translocation in the phloem and unloading in sink tissues, govern the partitioning of sugars in economically important organs, such as peach fruit. In this study, we took advantage of a symplastic tracer, carboxyfluorescein (CF), providing evidence for an apoplastic sucrose transfer in the early (SI) and middle (SIII) phases of peach fruit development. Moreover, using a combination of in situ hybridization and laser microdissection-assisted expression analysis, three putative sucrose transporters encoding genes (PpSUT1, PpSUT2, PpSUT4) were transcriptionally analyzed to relate their expression with sucrose storage in this organ. Our study revealed that PpSUT2 and PpSUT4 are the genes predominantly expressed in fruit flesh, and the detailed analysis of their expression pattern in the different cell types enabled us to suggest a specialized role in sucrose distribution. Both PpSUTs transporters could be involved in the retrieval of sucrose lost from the symplastic continuum of the phloem and, when expressed in parenchyma cells, they could be active in the import of sucrose into sink tissues, via symport from the apoplast. An alternative hypothesis has been proposed and discussed for PpSUT4 because of its putative tonoplastic localization. Taken together, our results provide new insights into the molecular mechanisms underpinning sucrose unloading and accumulation in peach fruit.