Ali Ferjani

Analysis of Leaf Development in fugu Mutants of Arabidopsis Reveals Three Compensation Modes That Modulate Cell Expansion in Determinate Organs

In multicellular organisms, the coordination of cell proliferation and expansion is fundamental for proper organogenesis, yet the molecular mechanisms involved in this coordination are largely unexplored. In plant leaves, the existence of this coordination is suggested by compensation, in which a decrease in cell number triggers an increase in mature cell size. To elucidate the mechanisms of compensation, we isolated five new Arabidopsis (Arabidopsis thaliana) mutants (fugu1–fugu5) that exhibit compensation. These mutants were characterized together with angustifolia3 (an3), erecta (er), and a KIP-RELATED PROTEIN2 (KRP2) overexpressor, which were previously reported to exhibit compensation. Time-course analyses of leaf development revealed that enhanced cell expansion in fugu2-1, fugu5-1, an3-4, and er-102 mutants is induced postmitotically, indicating that cell enlargement is not caused by the uncoupling of cell division from cell growth. In each of the mutants, either the rate or duration of cell expansion was selectively enhanced. In contrast, we found that enhanced cell expansion in KRP2 overexpressor occurs during cell proliferation. We further demonstrated that enhanced cell expansion occurs in cotyledons with dynamics similar to that in leaves. In contrast, cell expansion was not enhanced in roots even though they exhibit decreased cell numbers. Thus, compensation was confirmed to occur preferentially in determinate organs. Flow cytometric analyses revealed that increases in ploidy level are not always required to trigger compensation, suggesting that compensation is only partially mediated by ploidy-dependent processes. Our results suggest that compensation reflects an organ-wide coordination of cell proliferation and expansion in determinate organs, and involves at least three different expansion pathways.

Keep an Eye on PPi: The Vacuolar-Type H+-Pyrophosphatase Regulates Postgerminative Development in Arabidopsis

Oilseed germination requires gluconeogenesis, the conversion of storage lipids into carbohydrates, to sustain seedling heterotrophic growth. Pyrophosphate (PPi), a byproduct of ATP hydrolysis, is released by active metabolism in imbibed seeds. We report that the removal of PPi by plant vacuolar H+-pyrophosphatase is central for successful gluconeogenesis and resumption of postembryonic growth. Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H+-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1Pro:IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal β-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.

iTRAQ Analysis Reveals Mechanisms of Growth Defects due to Excess Zinc in Arabidopsis

The micronutrient zinc is essential for all living organisms, but it is toxic at high concentrations. Here, to understand the effects of excess zinc on plant cells, we performed an iTRAQ (for isobaric tags for relative and absolute quantification)-based quantitative proteomics approach to analyze microsomal proteins from Arabidopsis (Arabidopsis thaliana) roots. Our approach was sensitive enough to identify 521 proteins, including several membrane proteins. Among them, IRT1, an iron and zinc transporter, and FRO2, a ferric-chelate reductase, increased greatly in response to excess zinc. The expression of these two genes has been previously reported to increase under iron-deficient conditions. Indeed, the concentration of iron was significantly decreased in roots and shoots under excess zinc. Also, seven subunits of the vacuolar H+-ATPase (V-ATPase), a proton pump on the tonoplast and endosome, were identified, and three of them decreased significantly in response to excess zinc. In addition, excess zinc in the wild type decreased V-ATPase activity and length of roots and cells to levels comparable to those of the untreated de-etiolated3-1 mutant, which bears a mutation in V-ATPase subunit C. Interestingly, excess zinc led to the formation of branched and abnormally shaped root hairs, a phenotype that correlates with decreased levels of proteins of several root hair-defective mutants. Our results point out mechanisms of growth defects caused by excess zinc in which cross talk between iron and zinc homeostasis and V-ATPase activity might play a central role.

Identification of Zinc-Responsive Proteins in the Roots of Arabidopsis thaliana using a Highly Improved Method of Two-Dimensional Electrophoresis

Zinc (Zn) is an essential micronutrient for various physiological and metabolic processes in plants, although it is toxic in excess. To understand better Zn-responsive proteins, we developed a highly improved method of isoelectric focusing (IEF) in which whole lysate from Arabidopsis roots is subjected to IEF without any desalting steps. In this method, samples extracted with lysis buffer containing 1.5% SDS can also be directly applied to IEF. By applying this method to Zn-treated Arabidopsis roots, 10 up-regulated and 17 down-regulated proteins were identified, 15 of which showed a significant correlation with previously reported transcriptomic data.

The ATM-Dependent DNA Damage Response Acts as an Upstream Trigger for Compensation in the fas1 Mutation during Arabidopsis Leaf Development

The ATM-dependent DNA damage response acts as an upstream trigger for compensation, an enhanced cell expansion caused by defective cell proliferation, during Arabidopsis leaf development. During leaf development, a decrease in cell number often triggers an increase in cell size. This phenomenon, called compensation, suggests that some system coordinates cell proliferation and cell expansion, but how this is mediated at the molecular level is still unclear. The fugu2 mutants in Arabidopsis (Arabidopsis thaliana) exhibit typical compensation phenotypes. Here, we report that the FUGU2 gene encodes FASCIATA1 (FAS1), the p150 subunit of Chromatin Assembly Factor1. To uncover how the fas1 mutation induces compensation, we performed microarray analyses and found that many genes involved in the DNA damage response are up-regulated in fas1. Our genetic analysis further showed that activation of the DNA damage response and the accompanying decrease of cell number in fas1 depend on ATAXIA TELANGIECTASIA MUTATED (ATM) but not on ATM AND RAD3 RELATED. Kinematic analysis suggested that the delay in the cell cycle leads to a decrease in cell number in fas1 and that loss of ATM partially restores this phenotype. Consistently, both cell size phenotypes and high ploidy phenotypes of fas1 are also suppressed by atm, supporting that the ATM-dependent DNA damage response leads to these phenotypes. Altogether, these data suggest that the ATM-dependent DNA damage response acts as an upstream trigger in fas1 to delay the cell cycle and promote entry into the endocycle, resulting in compensated cell expansion.

Enhanced Cell Expansion in a KRP2 Overexpressor is Mediated by Increased V-ATPase Activity

Decreased cell numbers during leaf development often trigger increased cell size, a phenomenon called compensation. In compensation-exhibiting mutants, the unusually high cell expansion activity occurs through two different mechanisms during the post-mitotic stage of leaf development, except in the KIP-RELATED PROTEIN 2-overexpressing line (KRP2 o/e), whose cell sizes are 2-fold greater during proliferative growth. However, the molecular basis of compensated cell expansion (CCE) has not been characterized. The det3-1 mutant has a mutation in the C-subunit of the vacuolar-type H(+)-ATPase (V-ATPase) complex that causes a 50% decrease in its activity and cell size. To determine the contribution of V-ATPase activity to CCE, the cellular phenotypes of double mutants between det3-1 and compensation-exhibiting fugu5-1, an3-4, fas1-5 and KRP2 o/e were analyzed in detail. Interestingly, while decreased V-ATPase activity caused by det3-1 did not suppress CCE in fugu5-1, fas1-5 and an3-4, CCE in KRP2 o/e was totally suppressed. Furthermore, measurements revealed that the activity and quantity of the A-subunit of the V-ATPase complex were significantly increased in the shoots of KRP2 o/e plants. Importantly, the unusually increased size of actively dividing KRP2 o/e cells was restored to normal in the det3-1 background. Taken together, our data strongly suggest that CCE in KRP2 o/e, but not in other compensation-exhibiting mutants, occurs exclusively through the increase of V-ATPase activity.

Contribution of PPi-Hydrolyzing Function of Vacuolar H+-Pyrophosphatase in Vegetative Growth of Arabidopsis: Evidenced by Expression of Uncoupling Mutated Enzymes

The vacuolar-type H+-pyrophosphatase (H+-PPase) catalyzes a coupled reaction of pyrophosphate (PPi) hydrolysis and active proton translocation across the tonoplast. Overexpression of H+-PPase improves growth in various plant species, and loss-of-function mutants (fugu5s) of H+-PPase in Arabidopsis thaliana have post-germinative developmental defects. Here, to further clarify the physiological significance of this important enzyme, we newly generated three varieties of H+-PPase overexpressing lines with different levels of activity that we analyzed together with the loss-of-function mutant fugu5-3. The H+-PPase overexpressors exhibited enhanced activity of H+-PPase during vegetative growth, but no change in the activity of vacuolar H+-ATPase. Overexpressors with high enzymatic activity grew more vigorously with fresh weight increased by more than 24 and 44%, compared to the wild type and fugu5-3, respectively. Consistently, the overexpressors had larger rosette leaves and nearly 30% more cells in leaves than the wild type. When uncoupling mutated variants of H+-PPase, that could hydrolyze PPi but could not translocate protons, were introduced into the fugu5-3 mutant background, shoot growth defects recovered to the same levels as when a normal H+-PPase was introduced. Taken together, our findings clearly demonstrate that additional expression of H+-PPase improves plant growth by increasing cell number, predominantly as a consequence of the PPi-hydrolyzing activity of the enzyme.

Regulation of pyrophosphate levels by H+-PPase is central for proper resumption of early plant development.

The synthesis of DNA, RNA, and de novo proteins is fundamental for early development of the seedling after germination, but such processes release pyrophosphate (PPi) as a byproduct of ATP hydrolysis. The over-accumulation of the inhibitory metabolite PPi in the cytosol hinders these biosynthetic reactions. All living organisms possess ubiquitous enzymes collectively called inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of PPi into two orthophosphate (Pi) molecules. Defects in PPase activity cause severe developmental defects and/or growth arrest in several organisms. In higher plants, a proton-translocating vacuolar PPase (H+PPase) uses the energy of PPi hydrolysis to acidify the vacuole. However, the biological implications of PPi hydrolysis are vague due to the widespread belief that the major role of H+PPase in plants is vacuolar acidification. We have shown that the Arabidopsis fugu5 mutant phenotype, caused by a defect in H+PPase activity, is rescued by complementation with the yeast cytosolic PPase IPP1. In addition, our analyses have revealed that increased cytosolic PPi levels impair postgerminative development in fugu5 by inhibiting gluconeogenesis. This led us to the conclusion that the role of H+PPase as a proton-pump is negligible. Here, we present further evidence of the growth-boosting effects of removing PPi in later stages of plant vegetative development, and briefly discuss the biological role of PPases and their potential applications in different disciplines and in various organisms.

The Conflict Between Cell Proliferation and Expansion Primarily Affects Stem Organogenesis in Arabidopsis

Plant shoot organs such as stems, leaves and flowers are derived from specialized groups of stem cells organized at the shoot apical meristem (SAM). Organogenesis involves two major processes, namely cell proliferation and differentiation, whereby the former contributes to increasing the cell number and the latter involves substantial increases in cell volume through cell expansion. Co-ordination between the above processes in time and space is essential for proper organogenesis. To identify regulatory factors involved in proper organogenesis, heavy-ion beam-irradiated de-etiolated (det) 3-1 seeds have been used to identify striking phenotypes in the A#26-2; det3-1 mutant. In addition to the stunted plant stature mimicking det3-1, the A#26-2; det3-1 mutant exhibited stem thickening, increased floral organ number and a fruit shape reminiscent of clavata (clv) mutants. DNA sequencing analysis demonstrated that A#26-2; det3-1 harbors a mutation in the CLV3 gene. Importantly, A#26-2; det3-1 displayed cracks that randomly occurred on the main stem with a frequency of approximately 50%. Furthermore, the double mutants clv3-8 det3-1, clv1-4 det3-1 and clv2-1 det3-1 consistently showed stem cracks with frequencies of approximately 97, 38 and 35%, respectively. Cross-sections of stems further revealed an increase in vascular bundle number, cell number and size in the pith of clv3-8 det3-1 compared with det3-1. These findings suggest that the stem inner volume increase due to clv mutations exerts an outward mechanical stress; that in a det3-1 background (defective in cell expansion) resulted in cracking of the outermost layer of epidermal cells.

Regulation of PPi Levels Through the Vacuolar Membrane H+-Pyrophosphatase

Inorganic pyrophosphate (PPi) is a high-energy compound, although the free energy change of its hydrolysis is approximately 60 % that of ATP. PPi is generated as a by-product of macromolecule biosyntheses in plants, especially in proliferating cells. In living cells, the accumulation of PPi causes the suppression of these metabolic processes and the formation of insoluble Ca–PPi complexes. To avoid these negative effects, the vacuolar H+-pyrophosphatase (H+-PPase) hydrolyzes PPi and pumps H+ across the vacuolar membrane to maintain their acidic state. Importantly, recent studies on fugu5, the H+-PPase loss-of-function mutants, have clearly demonstrated that their phenotype is rescued by the expression of the yeast cytosolic PPase IPP1, which hydrolyzes cytosolic PPi but has no effect on vacuolar acidification, thus strongly suggesting that the role of the H+-PPase lies in the consumption of the inhibitory PPi rather than vacuolar acidification. In this chapter we describe the chemical properties and metabolic role of PPi, in addition to the physiological functions of H+-PPase and soluble PPase revealed by using several mutant lines.