Arturo Guevara-García

Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate.

Phosphorus (P) is one of the most important nutrients limiting agricultural production worldwide. In acid and alkaline soils, which make up over 70% of the worlds arable land, P forms insoluble compounds that are not available for plant use. To reduce P deficiencies and ensure plant productivity, nearly 30 million tons of P fertilizer are applied every year. Up to 80% of the applied P fertilizer is lost because it becomes immobile and unavailable for plant uptake. Therefore, the development of novel plant varieties more efficient in the use of P represents the best alternative to reduce the use of P fertilizers and achieve a more sustainable agriculture. We show here that the ability to use insoluble P compounds can be significantly enhanced by engineering plants to produce more organic acids. Our results show that when compared to the controls, citrate-overproducing plants yield more leaf and fruit biomass when grown under P-limiting conditions and require less P fertilizer to achieve optimal growth.

CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts

RNA editing changes the sequence of many transcripts in plant organelles, but little is known about the molecular mechanisms determining the specificity of the process. In this study, we have characterized CLB19 (also known as PDE247), a gene that is required for editing of two distinct chloroplast transcripts, rpoA and clpP. Loss-of-function clb19 mutants present a yellow phenotype with impaired chloroplast development and early seedling lethality under greenhouse conditions. Transcript patterns are profoundly affected in the mutant plants, with a pattern entirely consistent with a defect in activity of the plastid-encoded RNA polymerase. CLB19 encodes a pentatricopeptide repeat protein similar to the editing specificity factors CRR4 and CRR21, but, unlike them, is implicated in editing of two target sites.

Characterization of the Arabidopsis clb6 Mutant Illustrates the Importance of Posttranscriptional Regulation of the Methyl-d-Erythritol 4-Phosphate Pathway

The biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the two building blocks for isoprenoid biosynthesis, occurs by two independent pathways in plants. The mevalonic pathway operates in the cytoplasm, and the methyl-d-erythritol 4-phosphate (MEP) pathway operates in plastids. Plastidic isoprenoids play essential roles in plant growth and development. Plants must regulate the biosynthesis of isoprenoids to fulfill metabolic requirements in specific tissues and developmental conditions. The regulatory events that modulate the plant MEP pathway are not well understood. In this article, we demonstrate that the CHLOROPLAST BIOGENESIS6 (CLB6) gene, previously shown to be required for chloroplast development, encodes 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase, the last-acting enzyme of the MEP pathway. Comparative analysis of the expression levels of all MEP pathway gene transcripts and proteins in the clb6-1 mutant background revealed that posttranscriptional control modulates the levels of different proteins in this central pathway. Posttranscriptional regulation was also found during seedling development and during fosmidomycin inhibition of the pathway. Our results show that the first enzyme of the pathway, 1-deoxy-d-xylulose 5-phosphate synthase, is feedback regulated in response to the interruption of the flow of metabolites through the MEP pathway.

CHLOROPLAST BIOGENESIS Genes Act Cell and Noncell Autonomously in Early Chloroplast Development

In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis.

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.

A novel thioredoxin h is secreted in Nicotiana alata and reduces S-RNase in vitro.

Thioredoxins type h are classified into three subgroups. The subgroup II includes thioredoxins containing an N-terminal extension, the role of which is still unclear. Although thioredoxin secretion has been observed in animal cells, there is no evidence suggesting that any thioredoxin h is secreted in plants. In this study, we report that a thioredoxin h, subgroup II, from Nicotiana alata (NaTrxh) is secreted into the extracellular matrix of the stylar transmitting tract tissue. Fractionation studies showed that NaTrxh is extracted along with well characterized secretion proteins such as S-RNases and NaTTS (N. alata transmitting tissue-specific protein). Moreover, an NaTrxh-green fluorescent fusion protein transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana leaves was also secreted, showing that NaTrxh has the required information for its secretion. We performed reduction assays in vitro to identify potential extracellular targets of NaTrxh. We found that S-RNase is one of the several potential substrates of the NaTrxh in the extracellular matrix. In addition, we proved by affinity chromatography that NaTrxh specifically interacts with S-RNase. Our findings showed that NaTrxh is a new thioredoxin h in Nicotiana that is secreted as well as in animal systems. Because NaTrxh is localized in the extracellular matrix of the stylar transmitting tract and its specific interaction with S-RNase to reduce it in vitro, we suggest that this thioredoxin h may be involved either in general pollenpistil interaction processes or particularly in S-RNase-based self-incompatibility.

Arabidopsis thaliana mitogen-activated protein kinase 6 is involved in seed formation and modulation of primary and lateral root development

Arabidopsis MAP kinases are considered to have redundant functions. However, through a detailed phenotypic analysis, we demonstrated that MPK6 loss-of- function cause severe defects in embryo development, which are closed related with alterations in post-germination root development

A 42 bp fragment of the pmas1′ promoter containing an ocs-like element confers a developmental, wound- and chemically inducible expression pattern

Synthesis of mannopine in plant tissues infected with Agrobacterium tumefaciens is controlled by a divergent promoter (pmas2′ and pmas1′) that in 479 bp contains all the cis-acting elements necessary to direct tissue-specific and wound-inducible expression. In this report, using transgenic tobacco plants harboring a pmas1′-β-glucuronidase (GUS) gene fusion, we investigated the developmental expression pattern directed by pmas1′ in the early stages of development and the responses of pmas1′ to different chemical inducers. It was found that this promoter can respond to auxins, cytokinins, methyl jasmonate (MJ), salicylic acid (SA) and its analogue 2,6-dichloroisonicotinic acid (iNA). Treatment with chemical inducers also showed that the effects of iNA are organ-dependent, that wound-induction is a complex response mediated by at least two different chemical signals, and that MJ stimulates changes in the tissue-specific and developmental expression pattern directed by the pmas1′ promoter. Using chimeric promoters we demonstrate that an ocs-like element (ocs+1) directs MJ responses in an orientation-dependent manner and that sequences around the ocs+1 are important to maintain the inducible and developmental properties of this cis-regulatory element.

Proteomic analysis of chloroplast biogenesis (clb) mutants uncovers novel proteins potentially involved in the development of Arabidopsis thaliana chloroplasts

UNLABELLED Plant cells outstand for their ability to generate biomass from inorganic sources, this phenomenon takes place within the chloroplasts. The enzymatic machinery and developmental processes of chloroplasts have been subject of research for several decades, and this has resulted in the identification of a plethora of proteins that are essential for their development and function. Mutant lines for the genes that code for those proteins, often display pigment-accumulation defects (e.g., albino phenotypes). Here, we present a comparative proteomic analysis of four chloroplast-biogenesis affected mutants (cla1-1, clb2, clb5, clb19) aiming to identify novel proteins involved in the regulation of chloroplast development in Arabidopsis thaliana. We performed 2D-PAGE separation of the protein samples. These samples were then analyzed by computational processing of gel images in order to select protein spots with abundance shifts of at least twofold, statistically significant according to Students t-test (P<0.01). These spots were subjected to MALDI-TOF mass-spectrometry for protein identification. This process resulted in the discovery of three novel proteins potentially involved in the development of A. thaliana chloroplasts, as their associated mutant lines segregate pigment-deficient plants with abnormal chloroplasts, and altered mRNA accumulation of chloroplast-development marker genes. BIOLOGICAL SIGNIFICANCE This report highlights the potential of using a comparative proteomics strategy for the study of biological processes. Particularly, we compared the proteomes of wild-type seedlings and four mutant lines of A. thaliana affected in chloroplast biogenesis. From this proteomic analysis it was possible to detect common mechanisms in the mutants to respond to stress and cope with heterotrophy. Notably, it was possible to identify three novel proteins potentially involved in the development or functioning of chloroplasts, also it was demonstrated that plants annotated to carry T-DNA insertions in the cognate genes display pigment-deficient phenotypes, aberrant and underdeveloped chloroplasts, as well as altered mRNA accumulation of chloroplast biogenesis marker genes.

Data for a comparative proteomic analysis of chloroplast biogenesis (clb) mutants

This data article contains data related to the research article titled Proteomic analysis of chloroplast biogenesis (clb) mutants uncovers novel proteins potentially involved in the development ofArabidopsis thalianachloroplasts (de Luna-Valdez et al., 2014) [1]. This research article describes the 2-D PAGE-based proteomic analysis of wild-type and four mutant lines (cla1-1, clb2, clb5 and clb19) affected in the development of Arabidopsis thaliana chloroplasts. The report concludes with the discovery of three proteins potentially involved in chloroplast biogenesis. The information presented here represent the tables and figures that detail the processing of the raw data obtained from the image analysis of the 2-D PAGE gels.