Abdellatif Bahaji

Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production

Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. [(2009) Proc Natl Acad Sci USA 106:13124–13129], who showed that (i) SUS activity in wild type (WT) leaves is too low to account for normal rate of starch accumulation in Arabidopsis, and (ii) different organs of the sus1/sus2/sus3/sus4 SUS mutant impaired in SUS activity accumulate WT levels of ADP-glucose, UDP-glucose, cellulose and starch. However, these authors assayed SUS activity under unfavorable pH conditions for the reaction. By using favorable pH conditions for assaying SUS activity, in this work we show that SUS activity in the cleavage direction is sufficient to support normal rate of starch accumulation in WT leaves. We also demonstrate that sus1/sus2/sus3/sus4 leaves display WT SUS5 and SUS6 expression levels, whereas leaves of the sus5/sus6 mutant display WT SUS1–4 expression levels. Furthermore, we show that SUS activity in leaves and stems of the sus1/sus2/sus3/sus4 and sus5/sus6 plants is ∼85% of that of WT leaves, which can support normal cellulose and starch biosynthesis. The overall data disprove Barratt et al. (2009) claims, and are consistent with the possible involvement of SUS in cellulose and starch biosynthesis in Arabidopsis.

Enhancing Sucrose Synthase Activity in Transgenic Potato ( Solanum tuberosum L.) Tubers Results in Increased Levels of Starch, ADPglucose and UDPglucose and Total Yield

Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. To determine the impact of SuSy activity in starch metabolism and yield in potato (Solanum tuberosum L.) tubers we measured sugar levels and enzyme activities in tubers of SuSy-overexpressing potato plants grown in greenhouse and open field conditions. We also transcriptionally characterized tubers of SuSy-overexpressing and -antisensed potato plants. SuSy-overexpressing tubers exhibited a substantial increase in starch, UDPglucose and ADPglucose content when compared with controls. Tuber dry weight, starch content per plant and total yield of SuSy-overexpressing tubers increased significantly over those of control plants. In contrast, activities of enzymes directly involved in starch metabolism in SuSy-overexpressing tubers were normal when compared with controls. Transcriptomic analyses using POCI arrays and the MapMan software revealed that changes in SuSy activity affect the expression of genes involved in multiple biological processes, but not that of genes directly involved in starch metabolism. These analyses also revealed a reverse correlation between the expressions of acid invertase and SuSy-encoding genes, indicating that the balance between SuSy- and acid invertase-mediated sucrolytic pathways is a major determinant of starch accumulation in potato tubers. Results presented in this work show that SuSy strongly determines the intracellular levels of UDPglucose, ADPglucose and starch, and total yield in potato tubers. We also show that enhancement of SuSy activity represents a useful strategy for increasing starch accumulation and yield in potato tubers.

Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields

Structurally composed of the glucose homopolymers amylose and amylopectin, starch is the main storage carbohydrate in vascular plants, and is synthesized in the plastids of both photosynthetic and non-photosynthetic cells. Its abundance as a naturally occurring organic compound is surpassed only by cellulose, and represents both a cornerstone for human and animal nutrition and a feedstock for many non-food industrial applications including production of adhesives, biodegradable materials, and first-generation bioethanol. This review provides an update on the different proposed pathways of starch biosynthesis occurring in both autotrophic and heterotrophic organs, and provides emerging information about the networks regulating them and their interactions with the environment. Special emphasis is given to recent findings showing that volatile compounds emitted by microorganisms promote both growth and the accumulation of exceptionally high levels of starch in mono- and dicotyledonous plants. We also review how plant biotechnologists have attempted to use basic knowledge on starch metabolism for the rational design of genetic engineering traits aimed at increasing starch in annual crop species. Finally we present some potential biotechnological strategies for enhancing starch content.

Microbial Volatile Emissions Promote Accumulation of Exceptionally High Levels of Starch in Leaves in Mono-and Dicotyledonous Plants

Microbes emit volatile compounds that affect plant growth and development. However, little or nothing is known about how microbial emissions may affect primary carbohydrate metabolism in plants. In this work we explored the effect on leaf starch metabolism of volatiles released from different microbial species ranging from Gram-negative and Gram-positive bacteria to fungi. Surprisingly, we found that all microbial species tested (including plant pathogens and species not normally interacting with plants) emitted volatiles that strongly promoted starch accumulation in leaves of both mono- and dicotyledonous plants. Starch content in leaves of plants treated for 2 d with microbial volatiles was comparable with or even higher than that of reserve organs such as potato tubers. Transcriptome and enzyme activity analyses of potato leaves exposed to volatiles emitted by Alternaria alternata revealed that starch overaccumulation was accompanied by up-regulation of sucrose synthase, invertase inhibitors, starch synthase class III and IV, starch branching enzyme and glucose-6-phosphate transporter. This phenomenon, designated as MIVOISAP (microbial volatiles-induced starch accumulation process), was also accompanied by down-regulation of acid invertase, plastidial thioredoxins, starch breakdown enzymes, proteins involved in internal amino acid provision and less well defined mechanisms involving a bacterial- type stringent response. Treatment of potato leaves with fungal volatiles also resulted in enhanced levels of sucrose, ADPglucose, UDPglucose and 3-phosphoglycerate. MIVOISAP is independent of the presence of sucrose in the culture medium and is strongly repressed by cysteine supplementation. The discovery that microbial volatiles trigger starch accumulation enhancement in leaves constitutes an unreported mechanism for the elicidation of plant carbohydrate metabolism by microbes.

Post-Translational Redox Modification of ADP-Glucose Pyrophosphorylase in Response to Light is Not a Major Determinant of Fine Regulation of Transitory Starch Accumulation in Arabidopsis Leaves

ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme comprising two small and two large subunits that catalyze the production of ADP-glucose linked to starch biosynthesis. The current paradigm on leaf starch metabolism assumes that post-translational redox modification of AGP in response to light is a major determinant of fine regulation of transitory starch accumulation. According to this view, under oxidizing conditions occurring during the night the two AGP small subunits (APS1) are covalently linked via an intermolecular disulfide bridge that inactivates the protein, whereas under reducing conditions occurring during the day NADP-thioredoxin reductase C (NTRC)-dependent reductive monomerization of APS1 activates the enzyme. In this work we have analyzed changes in the redox status of APS1 during dark-light transition in leaves of plants cultured under different light intensities. Furthermore, we have carried out time-course analyses of starch content in ntrc mutants, and in aps1 mutants expressing the Escherichia coli redox-insensitive AGP (GlgC) in the chloroplast. We also characterized aps1 plants expressing a redox-insensitive, mutated APS1 (APS1mut) form in which the highly conserved Cys81 residue involved in the formation of the intermolecular disulfide bridge has been replaced by serine. We found that a very moderate, NTRC-dependent APS1 monomerization process in response to light occurred only when plants were cultured under photo-oxidative conditions. We also found that starch accumulation rates during the light in leaves of both ntrc mutants and GlgC-expressing aps1 mutants were similar to those of wild-type leaves. Furthermore, the pattern of starch accumulation during illumination in leaves of APS1mut-expressing aps1 mutants was similar to that of APS1-expressing aps1 mutants at any light intensity. The overall data demonstrate that post-translational redox modification of AGP in response to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis.

Enhancing the expression of starch synthase class IV results in increased levels of both transitory and long-term storage starch

Starch is an important renewable raw material with an increasing number of applications. Several attempts have been made to obtain plants that produce modified versions of starch or higher starch yield. Most of the approaches designed to increase the levels of starch have focused on the increment of the amount of ADP-glucose or ATP available for starch biosynthesis. In this work, we show that the overexpression of starch synthase class IV (SSIV) increases the levels of starch accumulated in the leaves of Arabidopsis by 30%-40%. In addition, SSIV-overexpressing lines display a higher rate of growth. The increase in starch content as a consequence of enhanced SSIV expression is also observed in long-term storage starch organs such as potato tubers. Overexpression of SSIV in potato leads to increased tuber starch content on a dry weight basis and to increased yield of starch production in terms of tons of starch/hectare. These results identify SSIV as one of the regulatory steps involved in the control of the amount of starch accumulated in plastids.

Microbial Volatile-Induced Accumulation of Exceptionally High Levels of Starch in Arabidopsis Leaves Is a Process Involving NTRC and Starch Synthase Classes III and IV

Microbial volatiles promote the accumulation of exceptionally high levels of starch in leaves. Time-course analyses of starch accumulation in Arabidopsis leaves exposed to fungal volatiles (FV) emitted by Alternaria alternata revealed that a microbial volatile-induced starch accumulation process (MIVOISAP) is due to stimulation of starch biosynthesis during illumination. The increase of starch content in illuminated leaves of FV-treated hy1/cry1, hy1/cry2, and hy1/cry1/cry2 Arabidopsis mutants was many-fold lower than that of wild-type (WT) leaves, indicating that MIVOISAP is subjected to photoreceptor-mediated control. This phenomenon was inhibited by cordycepin and accompanied by drastic changes in the Arabidopsis transcriptome. MIVOISAP was also accompanied by enhancement of the total 3-phosphoglycerate/Pi ratio, and a two- to threefold increase of the levels of the reduced form of ADP-glucose pyrophosphorylase. Using different Arabidopsis knockout mutants, we investigated the impact in MIVOISAP of downregulation of genes directly or indirectly related to starch metabolism. These analyses revealed that the magnitude of the FV-induced starch accumulation was low in mutants impaired in starch synthase (SS) classes III and IV and plastidial NADP-thioredoxin reductase C (NTRC). Thus, the overall data showed that Arabidopsis MIVOISAP involves a photocontrolled, transcriptionally and post-translationally regulated network wherein photoreceptor-, SSIII-, SSIV-, and NTRC-mediated changes in redox status of plastidial enzymes play important roles.

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Volatile compounds emitted by diverse phytopathogenic microorganisms promote plant growth and flowering through cytokinin action

It is known that volatile emissions from some beneficial rhizosphere microorganisms promote plant growth. Here we show that volatile compounds (VCs) emitted by phylogenetically diverse rhizosphere and non-rhizhosphere bacteria and fungi (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote growth and flowering of various plant species, including crops. In Arabidopsis plants exposed to VCs emitted by the phytopathogen Alternaria alternata, changes included enhancement of photosynthesis and accumulation of high levels of cytokinins (CKs) and sugars. Evidence obtained using transgenic Arabidopsis plants with altered CK status show that CKs play essential roles in this phenomenon, because growth and flowering responses to the VCs were reduced in mutants with CK-deficiency (35S:AtCKX1) or low receptor sensitivity (ahk2/3). Further, we demonstrate that the plant responses to fungal VCs are light-dependent. Transcriptomic analyses of Arabidopsis leaves exposed to A. alternata VCs revealed changes in the expression of light- and CK-responsive genes involved in photosynthesis, growth and flowering. Notably, many genes differentially expressed in plants treated with fungal VCs were also differentially expressed in plants exposed to VCs emitted by the plant growth promoting rhizobacterium Bacillus subtilis GB03, suggesting that plants react to microbial VCs through highly conserved regulatory mechanisms.

Phenotypic indicators of NaCl tolerance levels in rice seedlings: variations in development and leaf anatomy

The aim of this research is the early identification of distinctive responses to NaCl in rice cultivars that would indicate further stress-related effects in mature plants. For this purpose, we analysed some developmental and anatomical features in control and NaCl-stressed seedlings of two Japonica rice cultivars (Bomba and Bahia). Responses ascribed to osmotic stress were differentiated from those related to the ionic component of salinity by using in parallel a non-penetrating osmoticum (sorbitol). The general patterns of reduction in growth and variations in anatomical features of second leaf sections were similar in both cultivars. The main difference between them was the intensity of the response as a function of the stress agent. In general, the effect of NaCl was significantly stronger than that of sorbitol in cv. Bomba, whereas in cv. Bahia the effects of both stress agents were comparable. In this regard, the size of epidermal and bulliform cells as well as dimensions related to the vascular system, including xylem vessels, increased significantly in NaCl-stressed cv. Bomba leaves. This enlargement of xylem vessels agrees with the observed decrease in the rate of eosin transport and appears to be a distinctive anatomical indicator of NaCl sensitivity. The further impact of salinity on grain yield was proved to be stronger in cv. Bomba plants than in those of cv. Bahia.