Soluble invertase expression is an early target of drought stress during the critical,abortion-sensitive phase of young ovary development in maize

To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development.     

Functional reversion to identify controlling genes in multigenic responses: analysis of floral abortion

In many situations, organisms respond to stimuli by altering the activity of large numbers of genes. Among these, certain ones are likely to control the phenotype while others play a secondary role or are passively altered without directly affecting the phenotype. Identifying the controlling genes has proven difficult. However, in a few instances, it has been possible to reverse the phenotype by physiological or biochemical means without altering the genetics of the organism. During this functional reversion, only a few genes may respond, thus identifying those likely to be controlling the phenotype. Floral abortion during a water shortage in maize is an example because the response is inherently multigenic, and the phenotype can be reversed by physiological/biochemical means. A recent analysis used this reversal to reveal that only a few genes are likely to control the abortion phenotype. In maize, these genes coded for a cell wall invertase (Incw2), a soluble invertase (Ivr2), a ribosome-inactivating protein (RIP2), and phospholipase D (PLD1). The invertases appeared to control the normal sugar uptake by the ovaries. Their down-regulation depleted ovary sugar pools and resulted in an up-regulation of the genes for ribosome-inactivating protein and for phospholipase. The latter changes appeared to initiate senescence that degraded cell membranes, thus causing irreversible abortion. With these findings, these genes have become targets for preventing abortion. This approach might have value in other contexts with some additional methods.     

Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases

Two maize putative cell-wall invertase genes (Incw3 and Incw4) have been isolated by screening a genomic DNA library (Zea mays L. W22) using the cDNA probes encoding the two maize cell-wall invertases Incw1 and Incw2. The Incw3 and Incw4 genes contain six exons/five introns and five exons/four introns, respectively. The protein sequences deduced from both genes revealed a beta-fructosidase motif and a cysteine catalytic site known to be conserved in invertase genes. A detailed analysis of the protein and nucleotide sequences provides evidence that the Incw3 and the Incw4 genes encode putative cell-wall invertases. Furthermore, the isoelectric point deduced from the INCW4 protein sequence suggested that the Incw4 gene may encode a unique type of cell-wall invertase unbound in the apoplast. Gene expression studies using RT-PCR and in-situ RT-PCR hybridization showed that the Incw3 expression is organ/tissue-specific and developmentally regulated. In contrast, the Incw4 gene is constitutively expressed in all vegetative and reproductive tissues tested.     

A Similar Dichotomy of Sugar Modulation and Developmental Expression Affects Both Paths of Sucrose Metabolism: Evidence from a Maize Invertase Gene Family

Invertase and sucrose synthase catalyze the two known paths for the first step in carbon use by sucrose-importing plant cells. The hypothesis that sugar-modulated expression of these genes could provide a means of import adjustment was initially suggested based on data from sucrose synthases alone; however, this hypothesis remained largely conjectural without critical evidence for invertases. Toward this end, a family of maize invertases was cloned and characterized. Here, we show that invertases are indeed sugar modulated and, surprisingly, like the sucrose synthase genes, fall into two classes with contrasting sugar responses. In both families, one class of genes is upregulated by increasing carbohydrate supply (Sucrose synthase1 [Sus1] and Invertase2 [Ivr2]), whereas a second class in the same family is repressed by sugars and upregulated by depletion of this resource (Shrunken1 [Sh1] and Invertase1 [Ivr1]). The two classes also display differential expression during development, with sugar-enhanced genes (Sus1 and Ivr2) expressed in many importing organs and sugar-repressed, starvation-tolerant genes (Sh1 and Ivr1) upregulated primarily during reproductive development. Both the Ivr1 and Ivr2 invertase mRNAs are abundant in root tips, very young kernels, silk, anthers, and pollen, where a close relationship is evident between changes in message abundance and soluble invertase activity. During development, patterns of expression shift as assimilate partitioning changes from elongating silks to newly fertilized kernels. Together, the data support a model for integrating expression of genes differentially responsive to carbohydrate availability (i.e., feast and famine conditions) with developmental signals. The demonstration that similar regulatory patterns occur in both paths of sucrose metabolism indicates a potential to influence profoundly the adjustment of carbon resource allocation.     

The Miniature1 Seed Locus of Maize Encodes a Cell Wall Invertase Required for Normal Development of Endosperm and Maternal Cells in the Pedicel

Collective evidence demonstrates that the Miniature1 (Mn1) seed locus in maize encodes an endosperm-specific isozyme of cell wall Invertase, CWI-2. The evidence includes (1) isolation and characterization of ethyl methanesulfonate-induced mn1 mutants with altered enzyme activity and (2) a near-linear relationship between gene/dose and invertase activity and the CWI-2 protein. In addition, molecular analyses showed that the cDNA clone incw2 maps to the Mn1 locus and differentiates the six ethyl methanesulfonate-induced mn1 mutants of independent origin into two classes when RNA gel blot analyses were used. We also report two unexpected observations that provide significant new insight into the physiological role of invertase and its regulation in a developing seed. First, a large proportion of total enzyme activity (~90%) was dispensable (i.e., nonlimiting). However, below the threshold level of ~6% of wild-type activity, the endosperm enzyme controlled both the sink strength of the developing endosperm as well as the developmental stability of maternal cells in the pedicel in a rate-limiting manner. Our data also suggest an unusually tight coordinate control between the cell wall-bound and the soluble forms of invertase, which are most likely encoded by two separate genes, presumably through metabolic controls mediated by the sugars.     

Ivr2, a candidate gene for a QTL of vacuolar invertase activity in maize leaves. Gene-specific expression under water stress

Water shortage produced an early and large stimulation of acid- soluble invertase activity in adult maize leaves whereas cell wall invertase activity remained constant. This response was closely related to the mRNA level for only one of the invertase gene (Ivr2), encoding a vacuolar isoform. In parallel, four quantitative trait loci (QTLs) were detected for invertase activity under control and nine under stressful conditions. One QTL in control and one in stressed plants was located near to the lvr2 gene on chromosome 5. Other QTLs for invertase activity were found close to carbohydrate QTLs; some of them formed stress clusters.     

Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels

Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development.     

Sucrose and Nitrogen Supplies Regulate Growth of Maize Kernels

The growth of maize (Zea mays L.) kernels depends on the availabilityof carbon (C) and nitrogen (N) assimilates supplied by the motherplant and the capacity of the kernel to use them. Our objectiveswere to study the effects of N and sucrose supply levels ongrowth and metabolism of maize kernels. Kernel explants of Pioneer34RO6 were culturedin vitro with varying combinations of N (5to 30 m M) and sucrose (117 to 467 m M). Maximum kernel growthwas obtained with 10 m M N and 292 m M sucrose in the medium,and a deficiency of one assimilate could not be overcome bya sufficiency of the other. Increasing the N supply led to increasesin the kernel sink capacity (number of cells and starch granulesin the endosperm), activity of certain enzymes (soluble andbound invertases, sucrose synthase, and aspartate aminotransaminase),starch, and the levels of N compounds (total-N, soluble protein,and free amino acids), and decreased the levels of C metabolites(sucrose and reducing sugars). Conversely, increasing the sucrosesupply increased the level of endosperm C metabolites, freeamino acids, and ADPG-PPase and alanine transaminase activities,but decreased the activity of soluble invertase and concentrationsof soluble protein and total-N. Thus, while C and N are interdependentand essential for accumulation of maximum kernel weight, theyappear to regulate growth by different means. Nitrogen supplyaids the establishment of kernel sink capacity, and promotesactivity of enzymes relating to sucrose and nitrogen uptake,while sucrose regulates the activities of invertase and ADPG-PPase.Copyright 1999 Annals of Botany Company Zea mays, maize,, invertase, ADPG-PPase, media composition, sucrose, nitrogen, C/N.     

Gene expression studies on developing kernels of maize sucrose synthase (SuSy) mutants show evidence for a third SuSy gene

Previous studies have identified two tissue- and cell-specific, yet functionally redundant, sucrose synthase (SuSy) genes, Sh1 and Sus1, which encode biochemically similar isozymes, SH1 and SUS1 (previously referred to as SS1 and SS2, respectively). Here we report evidence for a third SuSy gene in maize, Sus3, which is more similar to dicot than to monocot SuSys. RNA and/or protein blot analyses on developing kernels and other tissues show evidence of expression of Sus3, although at the lowest steady-state levels of the three SuSy gene products and without a unique pattern of tissue specificity. Immunoblots of sh1sus1-1 embryos that are either lacking or deficient for the embryo-specific SUS1 protein have shown a protein band which we attribute to the Sus3 gene, and may contribute to the residual enzyme activity seen in embryos of the double mutant. We also studied developing seeds of the double mutant sh1sus1-1, which is missing 99.5% of SuSy enzyme activity, for evidence of co-regulation of several genes of sugar metabolism. We found a significant reduction in the steady-state levels of Miniature-1 encoded cell wall invertase2, and Sucrose transporter (Sut) mRNAs in the double mutant, relative to the lineage-related sh1Sus1 and sh1Sus1 kernels. Down-regulation of the Mn1 gene was also reflected in significant reductions in cell wall invertase activity. Co-regulatory changes were not seen in the expression of Sucrose phosphate synthase, UDP-glucose pyrophosphorylase, and ADP-glucose pyrophosphorylase.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.