Genetic evidence that invertase-mediated release of hexoses is critical for appropriate carbon partitioning and normal seed development in maize

 Cell wall-bound invertase (CWI) is spatially and temporally the first enzyme which metabolizes the incoming sucrose in developing seed of maize (Zea mays). Our previous studies have shown that the cell wall-bound invertase-2 (INCW2) isozyme encoded by the wild-type gene of the Miniature1 (Mn1) seed locus plays a critical role in seed development. Null mutations of the gene, such as the mn1 seed mutant which lacks invertase activity, are associated with a loss of ∼70–80% of the normal seed weight. We show here that under in vitro kernel culture conditions the hexose-based medium was similar to the sucrose-based medium in promoting the normal development of kernels of the Mn1, but not of the mutant mn1, genotype. Anatomical, biochemical, and immunohistological data showed that the mn1 kernels retain their mutant phenotype regardless of the presence of sucrose or hexoses in the culture media. The most drastic changes in the mn1 seed mutant were associated with a significant reduction in the size of the endosperm, but not in the pattern or the level of starch localization. Because Mn1 expression was temporally coincident with the endosperm cell divisions, INCW2 must play a critical role in providing hexose sugars for mitotic division, and only a minor role in generating carbon skeletal substrates for starch biosynthesis in the early stages of endosperm development. Furthermore, a lack of the wild-type seed phenotype of the mn1 mutant in hexose media suggests that a metabolic release of hexoses catalyzed by INCW2, rather than an exogenous source, is critical for both generating appropriate sugar-sensing signals for gene expression and for normal endosperm development. Received: 8 April 1998 / Accepted: 14 August 1998     

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.     

Miniature Seed6, encoding an endoplasmic reticulum signal peptidase,is critical in seed development

Endoplasmic reticulum (ER) type I signal peptidases (ER SPases I) are vital proteases that cleave signal peptides from secreted proteins. However, the specific function of ER SPase I in plants has not been genetically characterized, and the substrate is largely unknown. Here, we report the identification of a maize (Zea mays) miniature seed6 (mn6) mutant. The loss-of-function mn6 mutant exhibited severely reduced endosperm size. Map-based cloning and molecular characterization indicated that Mn6 is an S26-family ER SPase I, with Gly102 (box E) in Mn6 critical for protein function during processing. Mass spectrometric and immunoprecipitation analyses revealed that Mn6 is predominantly involved in processing carbohydrate synthesis-related proteins, including the cell wall invertase miniature seed1 (Mn1), which is specifically expressed in the basal endosperm transfer layer. RNA and protein expression levels of Mn1 were both significantly downregulated in the mn6 mutant. Due to the significant reduction in cell wall invertase activity in the transfer cell layer, mutation of Mn6 caused dramatic defects in endosperm development. These results suggest that proper maturation of Mn1 by Mn6 may be a crucial step for proper seed filling and maize development.

Miniature seed6 (Mn6), involved in maize (Zea mays) seed development, is necessary for processing the cell wall invertase Mn1, which is specifically expressed in the basal endosperm transfer layer.     

A Comparative Study on the Role of Cytokinins in Caryopsis Development in the Maize miniature1 Seed Mutant and Its Wild Type

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplication in developing maize caryopsis of a cell wall invertase-deficient miniature1 ( mn1 ) seed mutant and its wild type, Mn1 , genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mn1 compared with mn1 , resulting in a significantly higher cell number in the Mn1 endosperm. These data correlate with the previously reported peak levels of the Mn1 -encoded cell wall invertase-2 (INCW2) at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.     

The Maize Invertase-Deficient miniature-1 Seed Mutation Is Associated with Aberrant Pedicel and Endosperm Development

Genetic evidence is presented to show that the developmental stability of maternal cells in the pedicel at the base of maize seeds is determined by the genotype of the developing endosperm. An early degeneration and withdrawal of maternal cells from the endosperm of homozygous miniature (mn mn) seed mutants were arrested if mn plants were pollinated by the wild-type Mn pollen. Similarly, the stability of the wild-type, Mn mn, maternal cells was also dependent on whether or not these cells were associated with the normal (Mn) or the mutant (mn) endosperm on the same ear. Biochemical and cellular analyses indicated that developing mn kernels have extremely low (<0.5% of the wild type) to undetectable levels of both soluble and wall-bound invertase activities. Extracts from endosperm with a single copy of the Mn gene showed a significant increase in both forms of invertases, and we suggest it is the causal basis of the wild-type seed phenotype. Collectively, these data provide evidence that invertase-mediated maintenance of a physiological gradient of photosynthate between pedicel and endosperm constitutes the rate-limiting step in structural stability of maternal cells as well as normal development of endosperm and seed.     

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.     

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.     

Cell wall invertase-deficient miniature1 kernels have altered phytohormone levels

The Zea mays (maize) miniature1 (Mn1) locus encodes the cell wall invertase INCW2, which is localized predominantly in the basal endosperm transfer layer (BETL) of developing kernels and catalyzes conversion of sucrose into glucose and fructose. Mutations in Mn1 result in numerous changes that include a small kernel phenotype resulting from both decreased cell size and number. To explore the pleiotropic effects of this mutation, we investigated the levels of indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) in basal regions, upper regions, and embryos of developing kernels in the inbred line W22. We measured phytohormones from 6 to 28 days after pollination (DAP) in wild type (WT) and two alleles of mn1, mn1-1 and mn1-89. IAA was the predominant hormone in kernels, with WT levels of free IAA accumulating over time to more than 2microg/g of fresh weight. Kernels of mn1-1 accumulated up to 10-fold less IAA than WT, and levels of IAA sugar conjugates were similarly reduced. Although less abundant, differences were also observed in levels of ABA, JA, and SA between WT and the mn1 alleles. SA levels were increased by as much as 10-fold in mn1-1, and mn1-89 displayed intermediate SA levels at most timepoints. These findings indicate that invertase-mediated sucrose cleavage directly or indirectly regulates the levels of key plant hormones during seed development.     

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.     

Invertase Activity in Normal and Mutant Maize Endosperms during Development

A bound invertase and two soluble invertases are found in the developing endosperm of maize (Zea mays L.). The two soluble invertases can be separated on diethylaminoethyl-cellulose and Sephadex columns and distinguished by their kinetic constants. One soluble invertase, invertase I, is present from the 10- to 28-day stages of endosperm development with maximal activity per normal endosperm at the 12-day stage. In two endosperm mutant lines, shrunken-1 and shrunken-2, there is a second increase in invertase I activity later in development which could be a secondary effect caused by the abnormal metabolism in these lines. Another soluble invertase, invertase II, is present in the embryo upon germination and is also found in the very young developing endosperm (6-day stage). The third form of invertase, bound invertase, is present in the endosperm by the 6-day stage, and its activity remains approximately constant during development.