Josette Masle

The ERECTA gene regulates plant transpiration efficiency in Arabidopsis.

Assimilation of carbon by plants incurs water costs. In the many parts of the world where water is in short supply, plant transpiration efficiency, the ratio of carbon fixation to water loss, is critical to plant survival, crop yield and vegetation dynamics. When challenged by variations in their environment, plants often seem to coordinate photosynthesis and transpiration, but significant genetic variation in transpiration efficiency has been identified both between and within species. This has allowed plant breeders to develop effective selection programmes for the improved transpiration efficiency of crops, after it was demonstrated that carbon isotopic discrimination, Δ, of plant matter was a reliable and sensitive marker negatively related to variation in transpiration efficiency. However, little is known of the genetic controls of transpiration efficiency. Here we report the isolation of a gene that regulates transpiration efficiency, ERECTA. We show that ERECTA, a putative leucine-rich repeat receptor-like kinase (LRR-RLK) known for its effects on inflorescence development, is a major contributor to a locus for Δ on Arabidopsis chromosome 2. Mechanisms include, but are not limited to, effects on stomatal density, epidermal cell expansion, mesophyll cell proliferation and cell–cell contact.

Characterization of TCTP, the Translationally Controlled Tumor Protein, from Arabidopsis thaliana

The translationally controlled tumor protein (TCTP) is an important component of the TOR (target of rapamycin) signaling pathway, the major regulator of cell growth in animals and fungi. TCTP acts as the guanine nucleotide exchange factor of the Ras GTPase Rheb that controls TOR activity in Drosophila melanogaster. We therefore examined the role of Arabidopsis thaliana TCTP in planta. Plant TCTPs exhibit distinct sequence differences from nonplant homologs but share the key GTPase binding surface. Green fluorescent protein reporter lines show that Arabidopsis TCTP is expressed throughout plant tissues and developmental stages with increased expression in meristematic and expanding cells. Knockout of TCTP leads to a male gametophytic phenotype with normal pollen formation and germination but impaired pollen tube growth. Silencing of TCTP by RNA interference slows vegetative growth; leaf expansion is reduced because of smaller cell size, lateral root formation is reduced, and root hair development is impaired. Furthermore, these lines show decreased sensitivity to an exogenously applied auxin analog and have elevated levels of endogenous auxin. These results identify TCTP as an important regulator of growth in plants and imply a function of plant TCTP as a mediator of TOR activity similar to that known in nonplant systems.

Ethylene Modulates Root-Wave Responses in Arabidopsis

When stimulated to bend downward by being held at 45 degrees off vertical but unable to penetrate into agar-based media, Arabidopsis roots develop waving and looping growth patterns. Here, we demonstrate that ethylene modulates these responses. We determined that agar-containing plates sealed with low-porosity film generate abiotic ethylene concentrations of 0.1 to 0.3 μL L-1, whereas in plates wrapped with porous tape, ethylene remains at trace levels. We demonstrate that exogenous ethylene at concentrations as low as a few nanoliters per liter modulates root waving, root growth direction, and looping but through partly different mechanisms. Nutrients and Suc modify the effects of ethylene on root waving. Thus, ethylene had little effect on temporal wave frequency when nutrients were omitted but reduced it significantly on nutrient-supplemented agar. Suc masked the ethylene response. Ethylene consistently suppressed the normal tendency for roots of Landsberg erecta to skew to the right as they grow against hard-agar surfaces and also generated righthanded petiole twisting. Furthermore, ethylene suppressed root looping, a gravity-dependent growth response that was enhanced by high nutrient and Suc availability. Our work demonstrates that cell file twisting is not essential for root waving or skewing to occur. Differential flank growth accounted for both the extreme root waving on zero-nutrient plates and for root skewing. Root twisting was nutrient-dependent and was thus strongly associated with the looping response. The possible role of auxin transport in these responses and the involvement of circadian rhythms are discussed.

Functional Analysis of an Arabidopsis T-DNA “Knockout” of the High-Affinity NH4 + Transporter AtAMT1;1

NH4 + acquisition by plant roots is thought to involve members of the NH4 +transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH4 +. Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH4 + influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity 13NH4 +influx in the amt1;1:T-DNAmutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity 13NH4 +influx in the mutant by 30% compared with wild-type plants, whereas low-affinity 13NH4 + influx (250 μm–10 mm NH4 +) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 andAtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNAplants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH4 + and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH4 + transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1.

Effects of Ambient CO2 Concentration on Growth and Nitrogen Use in Tobacco (Nicotiana tabacum) Plants Transformed with an Antisense Gene to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

Growth of the R1 progeny of a tobacco plant (Nicotiana tabacum) transformed with an antisense gene to the small subunit of ribulose-1,5-carboxylase/oxygenase (Rubisco) was analyzed under 330 and 930 [mu]bar of CO2, at an irradiance of 1000 [mu]mol quanta m-2 s-1. Rubisco activity was reduced to 30 to 50% and 13 to 18% of that in the wild type when one and two copies of the antisense gene, respectively, were present in the genome, whereas null plants and wild-type plants had similar phenotypes. At 330 [mu]bar of CO2 all antisense plants were smaller than the wild type. There was no indication that Rubisco is present in excess in the wild type with respect to growth under high light. Raising ambient CO2 pressure to 930 [mu]bar caused plants with one copy of the DNA transferred from plasmid to plant genome to achieve the same size as the wild type at 330 [mu]bar, but plants with two copies remained smaller. Differences in final size were due mostly to early differences in relative rate of leaf area expansion (m2 m-2 d-1) or of biomass accumulation (g g-1 d-1): within less than 2 weeks after germination relative growth rates reached a steady-state value similar for all plants. Plants with greater carboxylation rates were characterized by a higher ratio of leaf carbon to leaf area, and at later stages, they were characterized also by a relatively greater allocation of structural and nonstructural carbon to roots versus leaves. However, these changes per se did not appear to be causing the long-term insensitivity of relative growth rates to variations in carboxylation rate. Nor was this insensitivity due to feedback inhibition of photosynthesis in leaves grown at high partial pressure of CO2 in the air (pa) or with high Rubisco activity, even when the amount of starch approached 40% of leaf dry weight. We propose that other intrinsic rate-limiting processes that are independent of carbohydrate supply were involved. Under plentiful nitrogen supply, reduction in the amount of nitrogen invested in Rubisco was more than compensated for by an increase in leaf nitrate. Nitrogen content of organic matter, excluding Rubisco, was unaffected by the antisense gene. In contrast, it was systematically lower at elevated pa than at normal pa. Combined with the positive effects of pa on growth, this resulted in the single-dose antisense plants growing as fast at 930 [mu]bar of CO2 as the wild-type plants at 330 [mu]bar of CO2 but at a lower organic nitrogen cost.

The jasmonic acid signaling pathway is linked to auxin homeostasis through the modulation of YUCCA8 and YUCCA9 gene expression

Interactions between phytohormones play important roles in the regulation of plant growth and development, but knowledge of the networks controlling hormonal relationships, such as between oxylipins and auxins, is just emerging. Here, we report the transcriptional regulation of two Arabidopsis YUCCA genes, YUC8 and YUC9, by oxylipins. Similar to previously characterized YUCCA family members, we show that both YUC8 and YUC9 are involved in auxin biosynthesis, as demonstrated by the increased auxin contents and auxin-dependent phenotypes displayed by gain-of-function mutants as well as the significantly decreased indole-3-acetic acid (IAA) levels in yuc8 and yuc8/9 knockout lines. Gene expression data obtained by qPCR analysis and microscopic examination of promoter-reporter lines reveal an oxylipin-mediated regulation of YUC9 expression that is dependent on the COI1 signal transduction pathway. In support of these findings, the roots of the analyzed yuc knockout mutants displayed a reduced response to methyl jasmonate (MeJA). The similar response of the yuc8 and yuc9 mutants to MeJA in cotyledons and hypocotyls suggests functional overlap of YUC8 and YUC9 in aerial tissues, while their function in roots shows some specificity, probably in part related to different spatio-temporal expression patterns of the two genes. These results provide evidence for an intimate functional relationship between oxylipin signaling and auxin homeostasis.

Over-expression of the rice OsAMT1-1 gene increases ammonium uptake and content, but impairs growth and development of plants under high ammonium nutrition

A transgenic approach was undertaken to investigate the role of a rice ammonium transporter (OsAMT1-1) in ammonium uptake and consequent ammonium assimilation under different nitrogen regimes. Transgenic lines overexpressing OsAMT1-1 were produced by Agrobacterium-mediated transformation of two rice cultivars, Taipei 309 and Jarrah, with an OsAMT1-1 cDNA gene construct driven by the maize ubiquitin promoter. Transcript levels of OsAMT1-1 in both Taipei 309 and Jarrah transgenic lines correlated positively with transgene copy number. Shoot and root biomass of some transgenic lines decreased during seedling and early vegetative stage compared to the wild type, especially when grown under high (2 mm) ammonium nutrition. Transgenic plants, particularly those of cv. Jarrah recovered in the mid-vegetative stage under high ammonium nutrition. Roots of the transgenic plants showed increased ammonium uptake and ammonium content. We conclude that the decreased biomass of the transgenic lines at early stages of growth might be caused by the accumulation of ammonium in the roots owing to the inability of ammonium assimilation to match the greater ammonium uptake.

Cell wall composition contributes to the control of transpiration efficiency in Arabidopsis thaliana

To identify loci in Arabidopsis involved in the control of transpirational water loss and transpiration efficiency (TE) we carried out an infrared thermal imaging-based screen. We report the identification of a new allele of the Arabidopsis CesA7 cellulose synthase locus designated AtCesA7(irx3-5) involved in the control of TE. Leaves of the AtCesA7(irx3-5) mutant are warmer than the wild type (WT). This is due to reduced stomatal pore widths brought about by guard cells that are significantly smaller than the WT. The xylem of the AtCesA7(irx3-5) mutant is also partially collapsed, and we suggest that the small guard cells in the mutant result from decreased water supply to the developing leaf. We used carbon isotope discrimination to show that TE is increased in AtCesA7(irx3-5) when compared with the WT. Our work identifies a new class of genes that affects TE and raises the possibility that other genes involved in cell wall biosynthesis will have an impact on water use efficiency.

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.

Biochemical Characterization of Two Wheat Phosphoethanolamine N-Methyltransferase Isoforms with Different Sensitivities to Inhibition by Phosphatidic Acid

In plants the triple methylation of phosphoethanolamine to phosphocholine catalyzed by phosphoethanolamine N-methyltransferase (PEAMT) is considered a rate-limiting step in the de novo synthesis of phosphatidylcholine. Besides being a major membrane phospholipid, phosphatidylcholine can be hydrolyzed into choline and phosphatidic acid. Phosphatidic acid is widely recognized as a second messenger in stress signaling, and choline can be oxidized within the chloroplast to yield the putative osmoprotectant glycine betaine. Here we describe the cloning and biochemical characterization of a second wheat PEAMT isoform that has a four times higher specific activity than the previously described WPEAMT/TaPEAMT1 enzyme and is less sensitive to product inhibition by S-adenosyl homocysteine, but more sensitive to inhibition by phosphocholine. Both enzymes follow a sequential random Bi Bi mechanism and show mixed-type product inhibition patterns with partial inhibition for TaPEAMT1 and a strong non-competitive component for TaPEAMT2. An induction of TaPEAMT protein expression and activity is observed after cold exposure, ahead of an increase in gene expression. Our results demonstrate direct repression of in vitro enzymatic activities by phosphatidic acid for both enzymes, with TaPEAMT1 being more sensitive than TaPEAMT2 in the physiological concentration range. Other lipid ligands identified in protein-lipid overlays are phosphoinositide mono- as well as some di-phosphates and cardiolipin. These results provide new insights into the complex regulatory circuits of phospholipid biosynthesis in plants and underline the importance of head group biosynthesis in adaptive stress responses.