Plants respond to heat stress by enhancing the expression of genes encoding heat shock protein (HSPs) genes through activation of heat shock factors (HSFs) which interact with heat shock elements present in the promoter of HSP genes. Plant HSFs have been divided into three conserved classes viz A, B and C. In the present study, a detailed analysis has been done of all rice HSFs, along with their spliced variants. Their chromosomal localization reveals that six HSFs are segmentally duplicated and four pairs of these segmentally duplicated HSF encoding genes show pseudo-functionalization. Expression profiling through microarray and quantitative real-time PCR showed that eight OsHsfs express at a higher level during seed development, while six HSFs are up-regulated in all the abiotic stresses studied. The expression of OsHsfA2a gene in particular was greatly stimulated by heat stress in both root and shoot tissues and also during panicle and seed development. OsHsfA3 was found more responsive to cold and drought stress, while OsHsfA7 and OsHsfA9 showed developing seed-specific expression. This study also revealed that spliced variants generally accumulated at a higher level in all the tissues examined. Different hormones/elicitors like ABA, brassinosteroids and salicylic acid also alter OsHsf gene expression. Calcium in combination with heat stress elevated further the level of HSF transcripts. Expression analysis by both microarray and real-time RT-PCR revealed a unique stable constitutive expression of OsHsfA1 across all the tissues and stresses. A detailed in silico analysis involving identification of unidentified domains has been done by MEME-motif tool in their full-length proteins as well as in DNA-binding domains. Analysis of 1 kb putative promoter region revealed presence of tissue-specific, abiotic stress and hormone-related cis-acting elements, correlating with expression under stress conditions.

Heat shock factors in rice (Oryza sativa L.): genome-wide expression analysis during reproductive development and abiotic stress.

Arabinogalactan proteins are widely distributed in plant tissues and cells, and may function in the growth and development of higher plants. To our knowledge, there is currently no direct evidence concerning the involvement of fasciclin-like arabinogalactan proteins (FLA) in sexual reproduction in Arabidopsis. In this study, Arabidopsis FLA3 was found to be specifically expressed in pollen grains and tubes. Subcellular localization showed that FLA3 anchors tightly to the plasma membrane, and its glycosylphosphatidylinositol anchor may affect its localization. FLA3-RNA interference transgenic plants had approximately 50% abnormal pollen grains (including shrunken and wrinkled phenotypes) which lacked viability. Cytological observations revealed that pollen abortion occurred during the transition from uninucleate microspores to bicellular pollens, with abnormal cellulose distribution seen by calcofluor white staining. Transmission electron microscopy showed that the basic structure of the exine layer in aberrant pollen was normal, but the intine layer appeared to have some abnormalities. Taken together, these results suggest that FLA3 is involved in microspore development and may affect pollen intine formation, possibly by participating in cellulose deposition. In FLA3-overexpressing transgenic plants, defective elongation of the stamen filament and reduced female fertility led to short siliques with low seed set, which suggested that ectopic expression of FLA3 in tissues may reduce or disrupt cell growth and then result in defects throughout the plant.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2010.04344.x

The fasciclin-like arabinogalactan protein gene, FLA3, is involved in microspore development of Arabidopsis

In this study we analyzed transcript abundance and promoters of genes encoding mitochondrial proteins to identify signaling pathways that regulate stress-induced gene expression. We used Arabidopsis (Arabidopsis thaliana) alternative oxidase AOX1a, external NADP H-dehydrogenase NDB2, and two additional highly stress-responsive genes, At2g21640 and BCS1. As a starting point, the promoter region of AOX1a was analyzed and functional analysis identified 10 cis-acting regulatory elements (CAREs), which played a role in response to treatment with H2O2, rotenone, or both. Six of these elements were also functional in the NDB2 promoter. The promoter region of At2g21640, previously defined as a hallmark of oxidative stress, shared two functional CAREs with AOX1a and was responsive to treatment with H2O2 but not rotenone. Microarray analysis further supported that signaling pathways induced by H2O2 and rotenone are not identical. The promoter of BCS1 was not responsive to H2O2 or rotenone, but highly responsive to salicylic acid (SA), whereas the promoters of AOX1a and NDB2 were unresponsive to SA. Analysis of transcript abundance of these genes in a variety of defense signaling mutants confirmed that BCS1 expression is regulated in a different manner compared to AOX1a, NDB2, and At2g21640. These mutants also revealed a pathway associated with programmed cell death that regulated AOX1a in a manner distinct from the other genes. Thus, at least three distinctive pathways regulate mitochondrial stress response at a transcriptional level, an SA-dependent pathway represented by BCS1, a second pathway that represents a convergence point for signals generated by H2O2 and rotenone on multiple CAREs, some of which are shared between responsive genes, and a third pathway that acts via EDS1 and PAD4 regulating only AOX1a. Furthermore, posttranscriptional regulation accounts for changes in transcript abundance by SA treatment for some genes.

http://www.plantphysiol.org/content/plantphysiol/147/4/1858.full.pdf

Identification of Regulatory Pathways Controlling Gene Expression of Stress-Responsive Mitochondrial Proteins in Arabidopsis

Plant β-1,3-glucanases are involved in plant defense and development. In rice (Oryza sativa), 14 genes encoding putative β-1,3-glucanases have been isolated and sequenced. However, only limited information is available on the function of these β-1,3-glucanase genes. In this study, we report a detailed functional characterization of one of these genes, Osg1. Osg1 encodes a glucanase carrying no C-terminal extension. Osg1 was found to be expressed throughout the plant and highly expressed in florets, leaf sheaths, and leaf blades. Investigations using real-time PCR, immunocytochemical analysis, and a GUS-reporter gene driven by the Osg1 promoter indicated that Osg1 was mainly expressed at the late meiosis, early microspore, and middle microspore stages in the florets. To elucidate the role of Osg1, we suppressed expression of the Osg1 gene by RNA interference in transgenic rice. The silencing of Osg1 resulted in male sterility. The pollen mother cells appeared to be normal in Osg1-RI plants, but callose degradation was disrupted around the microspores in the anther locules of the Osg1-RI plants at the early microspore stage. Consequently, the release of the young microspores into the anther locules was delayed, and the microspores began to degenerate later. These results provide evidence that Osg1 is essential for timely callose degradation in the process of tetrad dissolution.

A rice β-1,3-glucanase gene Osg1 is required for callose degradation in pollen development.

Wheat anther-specific invertase genes were haplotyped in wheat. Strong allelic selection occurred during wheat polyploidization, domestication and breeding because of their association with yield traits.
 Plant invertase hydrolyzes sucrose into glucose and fructose. Cell wall invertase (CWI), one of the three types of invertase, is essential for plant development. Based on isolated TaCWI genes from chromosomes 4A, 5B and 5D, two SNPs were detected in the promoter region of TaCWI-4A, and four SNPs and two Indels were present in the TaCWI-5D gene. No polymorphism was detected in TaCWI-5B coding or promoter regions. CAPS markers caps4A and caps5D were developed to discriminate haplotypes of TaCWI-4A and TaCWI-5D. Marker/trait association analysis indicated that Hap-5D-C at TaCWI-5D was significantly associated with higher thousand kernel weight (TKW) in 348 Chinese modern cultivars grown in multiple environments. Geographic distributions and changes over time of favored haplotypes showed that Hap-5D-C was the most frequent haplotype in modern cultivars and was strongly positively selected in six major wheat production regions worldwide. However, selection for haplotypes at TaCWI-4A was not so evident, possibly due to balancing effects of the two haplotypes on TKW and grain number per spike (GN). In rainfed production regions, Hap-4A-C was favored because it brought more seeds, but in well irrigated conditions, Hap-4A-T was favored in modern breeding because of higher TKW. Evolutionary analysis among wheat and its relatives showed that genetic diversity of TaCWI genes on chromosomes 4A and 5D declined dramatically in progression from the diploid level to modern polyploid cultivars. There was strong allelic selection during polyploidization, domestication and breeding.

A yield-associated gene TaCWI, in wheat: its function, selection and evolution in global breeding revealed by haplotype analysis

The tobacco gene g10 is preferentially and maximally expressed in mature pollen, shows homology to pectate lyases, and is the putative homologue of the tomato gene lat56. Analysis of regulatory elements within the g10 promoter was carried out to verify the importance of putative regulatory sequence motifs. Analysis of transgenic plants showed that 1190 bp of g10 5' sequence directed preferential expression of GUS in pollen, with bimodal peaks of expression just before and during pollen mitosis I, and in mature anthers. This was confirmed by northern analysis of native g10 transcripts in isolated spores. Transient expression analysis defined the minimal g10 promoter region capable of directing expression in pollen as -86 to +217. Three upstream regions within -427 bp modulate the expression from g10. Gain-of-function analyses showed that the region from -106 to -53 could enhance pollen-specific expression of a minimal CaMV 35S promoter. These analyses further showed that sequences upstream of -86 modulate expression in pollen, but are not essential for preferential pollen expression. The function of a conserved GTGA motif shared between the tobacco g10 and tomato lat56 promoters was demonstrated in g10. Thus, further functional evidence is provided for the conservation of mechanisms for the regulation of late pollen genes across species.

Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10.

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Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10.