QTLs and candidate genes for desiccation and abscisic acid content in maize kernels.

BACKGROUND: Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes. RESULTS: The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue. CONCLUSIONS: A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.

A joint transcriptomic, proteomic and metabolic analysis of maize endosperm development and starch filling.

The maize endosperm transcriptome was investigated through cDNA libraries developed at three characteristic stages: (i) lag phase [10 days after pollination (DAP)]; (ii) beginning of storage (14 DAP); and (iii) maximum starch accumulation rate (21 DAP). Expressed sequence tags for 711, 757 and 384 relevant clones, respectively, were obtained and checked manually. The proportion of sequences with no clear function decreased from 35% to 20%, and a large increase in storage protein sequences (i.e. 5% to 38%) was observed from stages (i) to (iii). The remaining major categories included metabolism (11%-13%), transcription-RNA processing-protein synthesis (13%-20%), protein destination (5%-9%), cellular communication (3%-9%) and cell rescue-defence (4%). Good agreement was generally found between category rank in the 10-DAP transcriptome and the recently reported 14-DAP proteome, except that kinases and proteins for RNA processing were not detected in the latter. In the metabolism category, the respiratory pathway transcripts represented the largest proportion (25%-37%), and showed a shift in favour of glycolysis at 21 DAP. At this stage, amino acid metabolism increased to 17%, whereas starch metabolism surprisingly decreased to 7%. A second experiment focused on carbohydrate metabolism by comparing gene expression at three levels (transcripts, proteins and enzyme activities) in relation to substrate or product from 10 to 40 DAP. Here, two distinct patterns were observed: invertases and hexoses were predominant at the beginning, whereas enzyme patterns in the starch pathway, at the three levels, anticipated and paralleled starch accumulation, suggesting that, in most cases, transcriptional control is responsible for the regulation of starch biosynthesis.

Developmental analysis of maize endosperm proteome suggests a pivotal role for pyruvate orthophosphate dikinase.

Although the morphological steps of maize (Zea mays) endosperm development are well described, very little is known concerning the coordinated accumulation of the numerous proteins involved. Here, we present a proteomic study of maize endosperm development. The accumulation pattern of 409 proteins at seven developmental stages was examined. Hierarchical clustering analysis allowed four main developmental profiles to be recognized. Comprehensive investigation of the functions associated with clusters resulted in a consistent picture of the developmental coordination of cellular processes. Early stages, devoted to cellularization, cell division, and cell wall deposition, corresponded to maximal expression of actin, tubulins, and cell organization proteins, of respiration metabolism (glycolysis and tricarboxylic acid cycle), and of protection against reactive oxygen species. An important protein turnover, which is likely associated with the switch from growth and differentiation to storage, was also suggested from the high amount of proteases. A relative increase of abundance of the glycolytic enzymes compared to tricarboxylic acid enzymes is consistent with the recent demonstration of anoxic conditions during starch accumulation in the endosperm. The specific late-stage accumulation of the pyruvate orthophosphate dikinase may suggest a critical role of this enzyme in the starch-protein balance through inorganic pyrophosphate-dependent restriction of ADP-glucose synthesis in addition to its usually reported influence on the alanine-aromatic amino acid synthesis balance.

QTLs for enzyme activities and soluble carbohydrates involved in starch accumulation during grain filling in maize.

ADPglucose, the essential substrate for starch synthesis, is synthesized in maize by a pathway involving at least invertases, sucrose synthase, and ADPglucose pyrophosphorylase, as shown by the starch-deficient mutants, mn1, sh1, and bt2 or sh2, respectively. To improve understanding of the relationship between early grain-filling traits and carbohydrate composition in mature grain, QTLs linked to soluble invertase, sucrose synthase, and ADPglucose pyrophosphorylase activities and to starch, sucrose, fructose, and glucose concentrations were investigated. In order to take into account the specific time-course of each enzyme activity during grain filling, sampling was carried out at three periods (15, 25, and 35 d after pollination) on 100 lines from a recombinant inbred family, grown in the field. The MQTL method associated with QTL interaction analysis revealed numerous QTLs for all traits, but only one QTL was consistently observed at the three sampling periods. Some chromosome zones were heavily labelled, forming clusters of QTLs. Numerous possible candidate genes of the starch synthetic pathway co-located with QTLs. Four QTLs were found close to the locus Sh1 (bin 9.01) coding for the sucrose synthase. In order to confirm the importance of this locus, the CAPS polymorphism of the Sh1 gene was analysed in 45 genetically unrelated maize lines from various geographical origins. The DNA polymorphism was significantly associated with phenotypic traits related to grain filling (starch and amylose content, grain matter, and ADPglucose pyrophosphorylase activity at 35 DAP). Thus, the Sh1 locus could provide a physiologically pertinent marker for maize selection.

A two-dimensional proteome map of maize endosperm.

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.

Regulation of vacuolar invertase by abscisic acid or glucose in leaves and roots from maize plantlets.

Recent studies have demonstrated in leaves of maize (Zea mays L.) plants submitted to a moderate water stress an early enhancement of vacuolar invertase activity that paralleled the expression of the vacuolar invertase Ivr2 gene and the accumulation of hexoses. In this paper, the direct role of abscisic acid (ABA) was checked by providing this hormone to the root medium of hydroponically grown maize plantlets. ABA supplied to 10-day-old seedlings appeared to enhance the vacuolar invertase activity within 1 h in roots and 2 h in leaves, the maximum being reached at 4 and 8 h, respectively. The Ivr2 gene expression varied accordingly, except that the maximum values were earlier. During the first 8 h of activity enhancement, hexose and sucrose concentrations were not significantly affected by ABA. The changes in activity were correlated to leaf and root ABA concentrations and they were concentration dependent in roots and leaves. In contrast, the addition of 1% glucose or polyethylene glycol, at the same osmotic potential, was ineffective on invertase activity, but glucose supply enhanced Ivr2 transcript levels, after 18 h, in a concentration-dependent manner in the leaf, whereas they were repressed at higher concentrations in intact roots. The latter result appeared specific to intact roots since similar treatments performed using excised leaf or root pieces confirmed a previous report on the enhancement of Ivr2 and Ivr1 transcript levels by glucose in roots [J. Xu et al. (1996) Plant Cell 8:1209-1220]. Therefore, ABA appears to be a strong inducer of Ivr2-invertase expression in roots and leaves.

The role of abscisic acid in the response of a specific vacuolar invertase to water stress in the adult maize leaf.

Among the numerous molecular and physiological modifications induced by water deficit, one of the earliest events observed in maize mature leaves subjected to water deprivation was a strong enhancement of acid vacuolar invertase activity, which occurred before the classical reduction in gas exchange due to stomatal closure. The increase in invertase activity coincided with the rapid accumulation of glucose and fructose that reached 8-fold the control leaf value. In addition, acid vacuolar invertase activity appeared to be highly correlated with xylem sap ABA concentration. In order to investigate the nature of the relationship between ABA and invertase activity, and to disconnect ABA from a likely sucrose side-effect, excised leaves were supplied with ABA or sucrose. As a consequence of ABA supply, a peak in leaf ABA appeared 4 h later which was followed by an enhancement of vacuolar invertase activity. ABA supply also produced a second maximum in leaf ABA. The transcript level of the Ivr2 gene encoding one vacuolar invertase presented the same two peaks pattern as leaf ABA, with a 2 h lag. This response was specific since the other invertase genes were not responding. Thus, ABA appeared to be a powerful enhancer of the IVR2 vacuolar invertase activity and expression. In the present conditions, the addition of sucrose had no effect on the enzyme activity.